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

193reduced. Summarizing, P. luminescens does produce a novel cell-cellsignaling molecule, PLAI-1, that controls the expression of the plu4568-plu4563 operon in a manner that is dependent on the orphan LuxR-likeregulator, PluR.RSV008-Hydroxyketone-mediated signal transduction in LegionellapneumophilaA. Kessler*, U. Schell, C. Harrison, H. HilbiMax von Pettenkofer Institute, LMU München, München, GermanyThe causative agent of Legionnaires' disease, Legionella pneumophila, is aparasite of environmental protozoa. L. pneumophila employs a biphasiclife cycle to replicate within and spread to new host cells. The switch fromthe replicative to the transmissive (virulent) state is coordinated by acomplex regulatory network, including signaling through endogenouslysynthesized small molecules (“autoinducers”) in a process termed “quorumsensing”. L. pneumophila produces and likely responds to -hydroxyketone signaling molecules [1].The lqs (Legionella quorum sensing) gene cluster harbors homologs of theVibrio cholerae cqsAS genes, i.e. lqsA and lqsS, flanking a gene calledlqsR. The autoinducer synthase LqsA catalyzes the production of LAI-1(Legionella autoinducer-1, 3-hydroxypentadecan-4-one), which ispresumably recognized by the sensor kinase LqsS, and LqsR is a novelputative response regulator that controls bacterial virulence andreplication. Functional studies and transcriptome analysis revealed thatlqsR, lqsS and lqsA regulate L. pneumophila-host cell interactions,extracellular filaments and a genomic “fitness island” [1].Through bioinformatic analysis an “orphan” homolog of the sensor kinaselqsS was identified in the L. pneumophila genome and termed lqsT. L.pneumophila lacking lqsT is impaired for virulence and intracellularreplication. Biochemical studies indicate that LqsS and LqsT are sensorkinases, which interact with the putative response regulator LqsR. Theseresults suggest that L. pneumophila responds to -hydroxyketone signalssensed by LqsS and LqsT, which converge on LqsR. Using biochemical,genetic and cellular microbial approaches, the role of LqsS, LqsT andLqsR in -hydroxyketone-mediated signal transduction and generegulation is further analyzed in detail.[1] Tiaden A., Spirig, T. and Hilbi, H. (2010) Bacterial gene regulation by -hydroxyketonesignaling. Trends Microbiol.18, 288-297.RSV009A high-frequency mutation in Bacillus subtilis: Requirements forthe decryptification of the gudB glutamate dehydrogenase geneF. Commichau*, S. Tholen, K. GunkaMicrobiology and Genetics, General Microbiology, Göttingen, GermanyCommon laboratory strains of Bacillus subtilis encode two catabolicglutamate dehydrogenases, the enzymatically active protein RocG and thecryptic enzyme GudB that is inactive due to a duplication of three aminoacids in its active centre (1, 2). The inactivation of the rocG gene results inpoor growth of the bacteria on complex media due to the accumulation oftoxic intermediates. Therefore, rocG mutants readily acquire suppressormutations that decryptify the gudB gene. We showed that thedecryptification occurs by a precise deletion of one part of the nine basepair direct repeat that causes the amino acid duplication. The deletionoccurs at the extremely high rate of 10 -4 (3). This is the highest mutationrate that was observed for a specific allele in B. subtilis. Mutationsaffecting the integrity of the direct repeat result in a strong reduction of themutation rate; however, the actual sequence of the repeat is not essential(3). We also demonstrated that the mutation rate of gudB is not affected bythe position of the gene on the chromosome. When the direct repeat wasplaced in the completely different context of an artificial promoter, theprecise deletion of one part of the repeat was also observed, but themutation rate was reduced by three orders of magnitude. Thus,transcription of the gudB gene seems to be essential for the high rate of theappearance of the gudB1 mutation. This idea is supported by the findingthat the transcription-repair coupling factor Mfd is required for thedecryptification of gudB. The Mfd-mediated coupling of transcription tomutagenesis can be regarded as a built-in precaution that facilitates theaccumulation of mutations preferentially in transcribed genes (4).1) Belitsky and Sonenshein, 1998. J. Bacteriol. 180: 6298-6305.2) Zeigler et al., 2008. J. Bacteriol. 190: 6983-6995.3) Gunka et al., 2012. J. Bacteriol. In press.4) Ayora et al., 1996. J. Mol. Biol. 256: 301-318.RSV010The PpsR Protein in Rhodospirillum rubrum: A majormetabolism coordinatorA. Carius*, L. Carius, H. GrammelMax Planck Institut, BMBF Nachwuchsgruppe Redoxphänomene inPurpurbakterien, Magdeburg, GermanyBeing a facultative anoxygenic photosynthetic bacterium, Rhodospirillumrubrum is able to adapt to various environmental conditions. Especially,the availability of oxygen demands for a precise regulatory response inpurple bacteria. Under anaerobic conditions anoxygenic photosynthesis isa very important energy source, but under aerobic conditions thephotosynthetic membranes (PM) can produce highly toxic reactive oxygenspecies. The switch between aerobic respiratory metabolism and anaerobicphotosynthetic metabolism is a great regulatory challenge for all purplebacteria so that many regulators are involved and several interestingstrategies have evolved.In most purple bacteria, two major regulatory systems, the RegB/RegAsystem and the PpsR system control photosynthetic gene expression. Bothsystems are well investigated in Rhodobacter species, often compared withR. rubrum. Basically, the RegB/RegA system activates photosyntheticgene expression when oxygen concentration is low, whereas PpsRrepresses the expression under aerobic conditions.Generally, in purple bacteria, the application of semiaerobic darkconditions results in basal expression of photosynthetic genes. However,so far only observed in R. rubrum, this PM-level can be strongly increasedby the addition of fructose or reduced glutathione to the culture medium(1). It is tempting to assume that both compounds provide additionalreducing equivalents thereby affecting identical redox regulatorypathways. Furthermore, no homologues of RegB/RegA can be found bygene sequence analysis in R. rubrum. This suggests a more central role ofthe PpsR-homologue in this bacterium.In this work, we show that PpsR in R. rubrum is most likely an activatorprotein for the photosynthetic genes. We used PpsR from heterologousexpression in E. coli for DNA-binding assays and overexpression strainsfor the elucidation of the role of PpsR in R. rubrum. The significance ofPpsR is underlined by the fact that no stable deletion strain could be created.1. Carius, A., M. Henkel, and H. Grammel. 2011. A glutathione redox effect on photosyntheticmembrane expression in Rhodospirillum rubrum. J. Bacteriol. 193(8):1893-1900.RSV011Fine-tuning of sulfur metabolism by a peptide-coding sRNA inthe photooxidative stress response of RhodobacterB. Berghoff*, Y. Hermanns, G. KlugJustus-Liebig-University, Microbiology and Molecular Biology, Gießen,GermanyThe photosynthetic model organism Rhodobacter sphaeroides facesphotooxidative stress due to the bacteriochlorophyll-mediated generation ofsinglet oxygen ( 1 O 2) in the light. In recent years several regulatory factors wereidentified which guide adaptation to these harmful conditions. The alternativesigma factor RpoE, which is on top of 1 O 2-dependent regulation, inducesamongst others the 219 nt long sRNA RSs0019 (1). RSs0019 contains a smallORF (150 nt), which was shown to be strongly translated under 1 O 2 stress.Overexpression of RSs0019 combined with microarray analysis suggested arole in fine-tuning of sulfur metabolism. To distinguish between peptide- versussRNA-driven effects, an RSs0019 variant with an internal stop-codon wasdesigned and compared to the genuine sRNA by real time RT-PCR. Theseexperiments both verified the microarray data and suggested RSs0019 to be abifunctional RNA. To gain further insights into the regulatory mechanisms,more precisely the RNA binding and potential translational effects of RSs0019,we made use of a lacZ-based in vivo reporter system for Rhodobacter. In thiscontext, mutational analyses of both RSs0019 and potential target mRNAs wereconstructed to uncover distinct interactions and outputs. Our data provideevidence that RSs0019 is a riboregulator which encodes a small peptide andfine-tunes the sulfur metabolism in Rhodobacter when sulfur stress originatesafter 1 O 2 generation.(1) Berghoff, B.A., Glaeser, J., Sharma, C.M., Vogel, J. and Klug, G. (2009) Photooxidative stressinducedand abundant small RNAs inRhodobacter sphaeroides.Mol. Microbiol., 74(6), 1497-1512.RSV012The conserved sRNA scr5239 controls DagA expression bytranslational repressionM. Vockenhuber*, B. SuessGoethe Universität Frankfurt, RNA Biochemie, Frankfurt, GermanyWe were interested in the identification and characterization of sRNAs inStreptomyces coelicolor. To find such transcripts we performed deepsequencing of the S. coelicolor transcriptome. That way we identified 63new non-coding RNAs and confirmed the expression for 11 [1].One sRNA - called scr5239 - found in the sequencing data especiallyattracted our interest because of its high degree of sequence and structureconservation. It is a 159 nt long sRNA with >90% conservation in 15streptomyces genomes and is constitutively expressed under mostBIOspektrum | Tagungsband 2012