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

195[4] Pustelny C, Albers A, Büldt-Karentzopoulos K, Parschat K, Chhabra SR, Cámara M, WilliamsP, Fetzner S (2009) Chem. Biol. 16:1259-1267RSP001Development of in vitro transcription system forCorynebacterium glutamicumJ. Holatko, R. Šilar, A. Rabatinova, H. Sanderova 1 , L. Krasny, J. Nasvera,M. Patek*Institute of Microbiology, Laboratory of Molecular Genetics of Bacteria,Prague 4, Czech RepublicIn vitro transcription analysis is a powerful tool to study various aspects oftranscriptional regulation of gene expression. We have developed the firstin vitro transcription system for Corynebacterium glutamicum, a producerof amino acids used in biotechnological processes. A bacterial RNApolymerase(RNAP) holoenzyme consists of a five-subunit ( 2, , ´, )core and a dissociable sigma subunit (factor), which is responsible for therecognition of specific promoter DNA sequences. The genome of C.glutamicum codes for 7 sigma subunits of RNAP. Most of the C.glutamicum promoters driving transcription of housekeeping genes arerecognized by RNAP with the primary sigma factor SigA. The primarylikefactor SigB is mainly involved in transcription of genes during thetransition phase between exponential and stationary growth phases. SigC,SigD, SigE, SigH and SigM of C. glutamicum are ECF (extracytoplasmicfunction) sigma factors involved in responses to various stress conditions(e.g. heat shock, oxidative and surface stresses). To determine, whichsigma factors are involved in expression of particular genes, we used thedeveloped in vitro transcription system for C. glutamicum. Thei n vitrotranscription system consists of RNAP holoenzyme reconstituted from thepurified His-tagged core RNAP and a separately isolated sigma factor.Plasmid pRLG770 constructs carrying promoters of various classes servedas templates. Using the in vitro analysis, RNAP+SigA recognizedspecifically the housekeeping promoters Pper from the C. glutamicumplasmid pGA1 and Pveg from Bacillus subtilis, whereas no transcriptionalactivity of RNAP+SigA on the SigH-dependent PdnaK promoter wasobserved. On the other hand, RNAP+SigH recognized specifically SigHdependentpromoters PdnaK and PsigB but not the housekeepingpromoters. Efficiency of the in vitro transcription system was optimizedusing various concentrations of RNAP and various ratios RNAP/sigmafactor. Analyses of further promoters recognized by isolated sigma factorsSigB, SigE and SigM are in progress.RSP002Identification and characterization of the LysR-typetranscriptional regulator HsdR for steroid-inducible expression ofthe 3-Hydroxysteroid dehydrogenase/carbonyl reductase gene inComamonas testosteroniG. Xiong*, W. Gong, E. MaserInstitute of Toxicology and Pharmacology, Klinikum, Uni. Kiel, Kiel, Germany3-hydroxysteroid dehydrogenase/carbonyl reductase (3-HSD/CR) fromComamonas testosteroni (C. testosteroni) is a key enzyme in steroiddegradation in soil and water. 3-HSD/CR gene (hsdA) expression can beinduced by steroids like testosterone and progesterone. Previously, wehave shown that induction of hsdA expression by steroids is a derepressionwhere steroidal inducers bind to two repressors, RepA and RepB, therebypreventing blocking of hsdA transcription and translation, respectively. Inthe present study, a new LysR-type transcriptional factor HsdR for 3-HSD/CR expression in C. testosteroni has been identified. The hsdR genelocates 2.58 kb downstream from hsdA on the C. testosteroni ATCC 11996chromosome with an orientation opposite to hsdA. The hsdR gene wascloned and recombinant HsdR protein was produced, as well as anti-HsdRpolyclonal antibodies. While heterologous transformation systems revealedthat HsdR activates the expression of hsdA gene, electrophoresis mobilityshift assays (EMSA) showed that HsdR specifically binds to the hsdApromoter region. Interestingly, the activity of HsdR is dependent ondecreased repression by RepA. Furthermore, in vitro binding assaysindicated that HsdR can contact with RNA polymerase. As expected, anhsdR knock-out mutant expressed low levels of 3-HSD/CR compared towild type C. testosteroni after testosterone induction. In conclusion, HsdRis a positive transcription factor for the hsdA gene and promote inductionof 3-HSD/CR expression in C. testosteroni.RSP003The transcriptional regulatory network of Corynebacterium jeikeiumK411 and its interaction with fatty acid degradation pathwaysH. Barzantny*, J. Schröder, I. Brune, A. TauchUniversität Bielefeld, Center for Biotechnology, Bielefeld, GermanyCorynebacterium jeikeium is a natural resident of the human skin and isnowadays frequently recognized as nosocomial pathogen in medicalfacilities. It causes severe infections in immunocompromised patients andthe treatment is often complicated by the comprehensive antibioticresistance of the organism 1,2 . The most prominent feature of C. jeikeium isits lipophilic lifestyle originating from the lack of a fatty acid synthasegene. Fatty acids are essential building blocks for cellular metabolites andmembrane or mycolic acid biosynthesis, since C. jeikeium is unable togrow solely on other carbon sources such as glucose or acetate 2 . Therefore,the organism encodes a large set of genes involved in -oxidation, whereofmost enzymatic functions relevant for the degradation of fatty acids areencoded by several paralogs 2 . Additionally, C. jeikeium encodes a uniquegene cluster that is potentially linked to fatty acid degradation 3 .To understand the transcriptional control of the essential pathway of -oxidation, the transcriptional regulatory network of the axilla isolate C.jeikeium K411 was reconstructed from the complete genome sequence.The current network reconstruction comprises 48 transcriptional regulatorsand 674 gene-regulatory interactions that can be assigned to fiveinterconnected functional modules. The analyses revealed that most genesinvolved in lipid degradation are under the combined control of the globalcAMP-sensing transcriptional regulator GlxR and the LuxR-familyregulator RamA, probably reflecting the essential role of lipid degradationin C. jeikeium.1 Funke G, von Graeventiz A, Clarridge III, JE and Bernard KA. 1997. Clinical microbiology of coryneformbacteria. Clin Microbiol Rev. 10:125-1592 Tauch A, Kaiser O, Hain T, Goesmann A, Weisshaar B, Albersmeier A, Bekel T, Bischoff N, Brune I,Chakraborty T, Kalinowski J, Meyer F, Rupp O, Schneiker S, Viehoever P and Pühler A. Complete genomesequence and analysis of the multiresistant nosocomial pathogenCorynebacterium jeikeiumK411, a lipidrequiringbacterium of the human skin flora. 2005. J Bacteriol. 187:4671-82.3Barzantny H, Brune I and Tauch A. Molecular basis of human body odour formation: insights deducedfrom corynebacterial genomes. 2011. Int J Cosmet Sci. doi: 10.1111/j.1468-2494.2011.00669.x. [Epubahead of print]RSP004Heterogeneity and timing in autoinducer regulated processesof Vibrio harveyiC. Anetzberger*, K. JungLMU Munich, Biology I, Planegg-Martinsried, GermanyBacteria produce and excrete signaling molecules, so called autoinducers(AIs), which allow them to monitor their population density and/or theirenvironment in a process best known as quorum sensing (QS). The marinebacterium Vibrio harveyi uses QS to regulate pathogenicity, biofilmformation, and bioluminescence. The bacterium synthesizes and respondsto three different classes of AIs, an acyl-homoserine lactone (HAI-1), afuranosylborate diester (AI-2) and a long-chain ketone (CAI-1).In order to understand how single cells behave within an AI activatedcommunity, AI induced processes were investigated in a homogeneousenvironment over time. Analysis of wild type single cells revealed thateven at high cell densities only 70% of the cells of a population producedbioluminescence. Moreover, fractionation of the population was found fortwo other AI controlled promoters of genes encoding virulence factors.These results indicate phenotypic heterogeneity of a genetic homogeneouspopulation. An artificial increase of the AI concentrations in the wild typeresulted in an all-bright population similarly to a luxO deletion mutant,which is AI independent. The capability of this mutant to produce biofilmwas significantly reduced. These data suggest that the non-differentiatingbacterium V. harveyi takes advantage of division of labor.In addition, results are provided for the temporal variation of theextracellular AI concentrations over time. AI concentrations and QSregulated functions of V. harveyi were monitored simultaneously in agrowing culture. In the early exponential growth phase only AI-2 wasdetectable and bioluminescence was induced. In the exponential growthphase both HAI-1 and AI-2 reached their maximum values,bioluminescence further increased and exoproteolytic activity wasinduced. In the stationary growth phase HAI-1 and AI-2 were adjusted toequal concentrations, exoproteolytic activity reached its maximum, andCAI-1 was detectable. Furthermore, formation of a stable and maturebiofilm was dependent on a correct timing of HAI-1 and AI-2concentrations. Our results demonstrate that not the cell density per se isimportant, but that AIs rather control the development of a V. harveyipopulation.RSP005Role of the small RNA RSs2430 in the regulation ofphotosynthesis genes in Rhodobacter sphaeroidesN. Mank*, B. Berghoff, Y. Hermanns, G. KlugInstitut f. Mikro- und Molekularbiologie, Klug, Gießen, GermanySmall RNAs (sRNAs) play a regulatory role in the adaptation of variousbacteria to changing environmental conditions. The identification ofsRNAs, using RNA-seq based on 454 pyrosequencing, in the phototrophicbacterium Rhodobacter sphaeroides (1) was of major interest because ofits high metabolic versatility. In particular, synthesis of the photosyntheticapparatus is regulated in an oxygen- and light-dependent manner. In aphysiological screen the sRNA RSs2430 was also found to be influencedby the oxygen tension. Induction of RSs2430 depends on the PrrB/PrrAsystem, which is a major regulatory system for redox control ofphotosynthesis genes. Here we present how overexpression and knockBIOspektrum | Tagungsband 2012