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

RGP022Identification and Characterization of small RNAs inAgrobacterium tumefaciensA. Overlöper* 1 , I. Wilms 1 , B. Voss 2 , W. Hess 2 , C. Sharma 3 , J. Vogel 3 ,F. Narberhaus 11 Department of Biology of Microorganisms, Ruhr-University, Bochum,Germany2 Institute of Biology III, Albert-Ludwigs-University, Freiburg, Germany3 Institute for Molecular Infection Biology, Julius-Maximilians-University,Würzburg, GermanyIn the past years small noncoding RNAs (sRNAs) have received enormousattention as a new class of gene expression regulators. The largest and mostextensively studied set of sRNAs act through base pairing with target RNAs,usually modulating the translation and stability of mRNAs (1).Using a comparative bioinformatic approach (2) we identified diversesRNAs in the plant pathogen Agrobacterium tumefaciens. Two tandemsRNAs control the expression of at least three ABC transporters amongthem the periplasmic binding protein of the GABA transporter. Themolecular details of the sRNA-mRNA interaction will be presented.By using a differential RNA sequencing (dRNA-seq) technology (3) wediscovered many new sRNA candidates on all four A. tumefaciens replicons,the circular chromosome, the linear chromosome, the At-plasmid and the Tiplasmid.At least one sRNA is highly induced under virulence conditions.[1] Waters, L. S. and G. Storz, (2009): Regulatory RNAs in bacteria. Cell 136: 615-628.[2] Axmann, I.M. et al (2005): Identification of cyanobacterial non-coding RNAs by comparativegenome analysis. Genome Biol 6: R73.[3] Sharma, C.M. et al (2010): The primary transcriptome of the major human pathogen Helicobacterpylori. Nature 464: 250-255.RGP023FrlR, a novel transcription factor that strongly regulatesthe catabolic frl-operon in B. subtilis 168S. Klatte* 1,2 , V. Deppe 1,2 , J. Bongearts 1 , K.-H. Maurer 1 , F. Meinhardt 21 Henkel AG & Co. KGaA, Biotechnology, Düsseldorf, Germany2 Institute for Molecular Microbiology and Biotechnology, WestphalianWilhelms-University, Münster, GermanyThe Gram-positive model organism Bacillus subtilis metabolizes the carbonandnitrogen source Amadori product that occurs in soil and long storedfood. Amadori products are. CodY, a global transcription regulator in Grampositivebacteria, was shown to regulate the promoter upstream of the frlBgene [2] . In this study, the transcriptional regulator named FrlR wasinvestigated which is a GntR-type transcription factor and also represses theexpression of frlBONMD [3] . Its gene is located downstream of thefrlBONMD operon and is inversely orientated to them. Electrophoreticmobility shift assays revealed a total of three FrlR binding sites within thefrlBONMD-frlR region. The regulator protein binds to the promoter P frlB, thefirst intergenic region of the operon and the promoter P frlR. From theseregions a GntR binding motif 5´-(N) yGT.N 2.TA.N 2.AC(N) y-3´ was derived.However, the frl-operon is regulated by CodY and FrlR together becausethey bind at P frlB, simultaneously. Remarkably, the intergenic region of frlBand frlO genes contains a 38 bp perfect palindrome in which the FrlRbinding site is located. By this, FrlR causes repression of the downstreamgenes. Additionally, first experiments indicate a negative effect on thetranscription of the downstream located genes by the palindrome itself.[1] Wiame et al (2002): J. Biol. Chem. 277:42523-42529.[2] Belitsky et al (2008): J. Bacteriol. 190:1224-1236.[3] Deppe et al submitted.RGP024pH-dependent expression of the alsSD Operon of B.subtilis and regulation by AlsRC. Frädrich*, A. Hartmann, E. HärtigInstitute of Microbiology, University of Technology, Braunschweig,GermanyBacillus subtilis forms Acetoin under anaerobic fermentative growthconditions. It requires acetolactate synthase and -decarbobxylase encoded bythe alsSD operon. The alsSD expression is induced by addition of acetate tothe growth medium, low pH and aerobic stationary phase. The regulatorAlsR is essential for alsS-lacZ reportergene expression under all growthconditions tested. The AlsR regulator is a member of the LysR-typetranscriptional regulators (LTTR) and composed of two domains: an N-terminal DNA binding domain with a winged HTH motif and a C-terminalregulatory domain. Most regulators of the LysR family are activated bybinding of an inducer to the regulatory domain. For AlsR acetate or areduced pH is postulated as inducing signal.We measured alsS-lacZ expression under different pH conditions and in thepresence of various organic acids to discriminate between reduced pH oraccumulation of organic acids like acetate as inducing signal. In addition weperformed in vitro DNA-binding studies with pH values from 5 to 9 toanalyze AlsR binding.In order to identify functional relevant amino acid residues of the effectordomain we mutagenized the alsR gene and tested the in vivo activity ofmutant AlsR proteins in an in vivo complementation system. Here, mutatedalsR genes were integrated into the amyE locus of a B. subtilis alsR knockout mutant strain and were expressed under the control of the xyloseinduciblexylA promoter. AlsR activity was monitored by ß-galactosidaseactivities deriving from the AlsR-dependent alsS-lacZ reporter gene fusion.RGP025A specialized FMN riboswitch confers roseoflavinresistance to Streptomyces davawensisD.B. Pedrolli*, M. MackInstitute of Technical Microbiology, Mannheim University of AppliedSciences, Mannheim, GermanyThe expression of bacterial genes involved in riboflavin production andtransport are regulated by FMN riboswitches present in the 5’-untranslatedregions of the corresponding mRNAs. The aptamer portion of the FMNriboswitches binds FMN (flavin mononucleotide, the phosphorylatedderivative of riboflavin) and regulates gene expression in combination withan expression platform either by transcription termination or by preventingtranslation initiation. Streptomyces davawensis synthesizes the antibioticroseoflavin, which is toxic to gram-positive but also to gram-negativebacteria if the compound is able to enter the cell. Roseoflavin isphosphorylated to roseoflavin mononucleotide (RoFMN) whichsubsequently is adenylated to roseoflavin adenine dinucleotide (RoFAD).RoFMN and RoFAD may inactivate flavoenzymes. In addition, bacterialFMN riboswitches were found to be targets for roseoflavin/RoFMN. S.davawensis, in contrast to Bacillus subtilis or Streptomyces coelicolor, isroseoflavin resistant. Our hypothesis was that S. davawensis contained aspecialized FMN riboswitch, which is not affected by RoFMN. To test this,plasmids were constructed, which contained the FMN riboswitches from B.subtilis, S. coelicolor and S. davawensis directly downstream of the T7promoter and upstream of the firefly luciferase reporter gene. The plasmidswere used for an in vitro transcription/translation reaction (TK/TL) in thepresence of FMN or RoFMN. RoFMN, which was not commerciallyavailable, was produced by human flavokinase. A strong reduction of theluciferase reporter activity was found in the TK/TL in the presence of FMN,which suggests that less of the reporter enzyme was produced. Apparently,the FMN riboswitches of the three bacteria responded similarly to FMN.Upon addition of RoFMN in the TK/TL, the luciferase activity was reducedin the assays containing the FMN riboswitches from the roseoflavinsensitive organisms B. subtilis and S. coelicolor. In the correspondingTK/TL containing the S. davawensis FMN riboswitch, however, theluciferase activity was not reduced in the presence of RoFMN. Based on theknown ability of the flavokinase/FAD synthetase from S davawensis toconvert roseoflavin into RoFMN, we conclude that the FMN riboswitchfrom this bacterium is specialized to not respond to RoFMN. Subsequent invivo studies are necessary to confirm this finding.[1] Serganov, A. et al (2009): Nature 458, 233-237.[2] Grill, S. et al (2008): J Bacteriol 190, 1546-1553.RGP026Regulation of translation in halophilic archaeaK. Gäbel*, O. Hering, J. SoppaInstitute für Molecular Bio Science, Goethe-University, Frankfurt am Main,GermanyTranslation is a very important step for the expression of genetic informationinto the phenotypes of cells or organisms. Regulation of translation typicallyoccurs during initiation because this step is rate-limiting. Three differentmechanisms for translation initiation were shown to operate in haloarchaea.About 2/3 of the transcripts are leaderless. Surprisingly most leaderedtranscripts are devoid of a Shine Dalgarno (SD) motif and it was shown thatspektrum | Tagungsband 2011