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

a novel initiation mechanism operates on these transcripts [1]. At very lowfrequency transcripts with SD motifs also exist, therefore three differentinitiation mechanisms operate in haloarchaea simultaneously [2]. To analyzethese mechanisms transcripts containing three different initiation sites infront of the dhfr reporter gene were generated and it was verified that allthree initiation sites operate in vivo. This enables the characterization of thedifferential usage of the mechanisms under various conditions, i.e. indifferent media with various C-sources, N-sources and salt concentrations orat different temperatures. Quantification of the protein levels by Westernblots and the transcript levels by Northern blots allowed determination oftranslational efficiencies. We could reveal that the initiation mechanisms areused differentially under specific conditions. These results show that H.volcanii applies the three different initiation mechanisms for conditionalregulation of translational efficiencies and thus uses translational regulons toadapt to changing environmental conditions.[1] Brenneis, M. et al (2007): PLoS Genet. 3(12): e229.[2] Hering, O. et al (2009): Mol. Microbiol. 71:1451-1463.RGP027Light-dependent gene induction in Aspergillus nidulansrequires release of the repressor LreA and binding of theactivator FphAM. Hedtke*, J. Rodriguez-Romero, R. FischerDepartment of Microbiology, Karlsruhe Institute of Technology (KIT),Karlsruhe, GermanyLight serves as an important environmental signal to regulate developmentand metabolism in many fungi and has been studied to some detail inNeurospora crassa and Aspergillus nidulans. A. nidulans develops mainlyasexually in the light and sexually in the dark. The red-light sensorphytochrome (FphA) and the WC-1 homologue blue-light receptor LreAhave been shown to mediate the light response in A. nidulans [1]. There isevidence that both proteins form a light regulator complex (LRC). LreB(WC-2) and VeA are probably also components of this complex [2].Using Chromatin-Immunoprecipitation (ChIP) and quantitative Real TimePCR we show that HA-tagged FphA and LreA bind to the promoters of theA. nidulans homologues of N. crassa con-10 (conJ) and ccg-1 (ccgA). In A.nidulans conJ and ccgA are both induced during development but are alsostrongly upregulated in hyphae after short exposure to light.Surprisingly we found LreA bound to the conJ and ccgA promoter only inthe dark probably acting as a repressor. In contrast, FphA is recruited to thepromoters after short illumination and seems to function as activator oftranscription. These results suggest that the LRC is not a tight proteincomplex but rather transient and that light induction depends onderepression followed by induction through FphA.[1] Blumenstein, A. et al (2005): Curr. Biol. 15(20):1833-8.[2] Purschwitz, J. et al (2008): Mol. Genet. Genomics 18(4):255-9.RGP028Effect of primary metabolism on secondary metaboliteproduction in Aspergillus terreusM. Greßler* 1 , C. Zähle 2 , K. Scherlach 2 , C. Hertweck 2 , M. Brock 11 Junior Research Group Microbial Biochemistry and Physiology, HansKnöll Institute (HKI), Jena, Germany2 Biomolecular Chemistry, Hans Knöll Institute (HKI), Jena, GermanyGenome sequencing has shown that Aspergillus terreus has the potential toproduce a great variety of different natural products. Although severalmetabolites have been identified, it can be assumed that the potential toproduce secondary metabolites is much larger than currently known. Severalstrategies have been developed to discover new metabolites produced byfilamentous fungi. Besides the use of epigenetic modifiers or co-cultivationexperiments, targeted overexpression of putative transcription factorsprovides a promising tool to activate silent gene clusters. Here, weinvestigated the expression of the only complete PKS-NRPS hybrid genepresent in the genome of A. terreus. Since overexpression of a putativetranscriptional activator adjacent to the PKS-NRPS gene did not activategene transcription, we constructed a lacZ reporter strain to screen fornaturally inducing conditions. Results revealed that expression was activatedin the presence of several amino acids and enhanced by alkaline pH.However, glucose mediated carbon catabolite repression remained as thedominating inhibiting factor. When the adjacent transcription factor, whichfailed to induce PKS-NRPS expression in initial experiments, was expressedunder naturally non-inducing, but also non-repressing conditions, activationof the PKS-NRPS gene was observed. Thus, factors involved in regulationof primary metabolism can override activating effects from cluster specifictranscription factors. Finally, product identification revealed that the genecluster is responsible for producing metabolites of the fruit rot toxin family.RGP029Analysis of DNA binding by Qdr1 and Qdr2, twotranscriptional regulators of quinaldine degradation byArthrobacter nitroguajacolicus Rü61aH. Niewerth*, K. Parschat, S. FetznerInstitute for Molecular Microbiology and Biotechnology (IMMB),Westphalian Wilhelms-University, Münster, GermanyArthrobacter nitroguajacolicus Rü61a is able to utilize quinaldine as sourceof carbon and energy. The genes that enable A. nitroguajacolicus to convertquinaldine to anthranilate are clustered in two „upper pathway” operonswhich are localized on the 113 kbp linear plasmid pAL1. A third operonlocated downstream of the „upper pathway” operons codes for anthranilateconversion via CoA-thioester intermediates [1].Qdr1 and Qdr2, two PaaX-like DNA binding proteins encoded by pAL1, areinvolved in the regulation of the utilization of quinaldine. The canonicalPaaX repressors use phenylacetyl-CoA as effector and are known totranscriptionally regulate the phenylacetate catabolon of E. coli [2] andPseudomonas putida [3]. Electrophoretic mobility shift assays withrecombinant Qdr1 and Qdr2 showed that both regulators bind specifically tothe promoter regions of all three operons, and revealed that the dissociationof Qdr-DNA complexes is induced by anthraniloyl-CoA.The transcriptional start points of qdr1 and qdr2 were identified by 5´RACE(rapid amplification of 5´cDNA ends) analysis. The deduced promoterregions of qdr1 and qdr2 bear a strong resemblance to the -10 and -35 regionof the σ 32 promoter sequence of E. coli. The interaction of each regulatorwith these promoters is currently being studied by gel shift analysis. TheDNA sequences recognized by Qdr1 and Qdr2 will be identified by DNase Ifootprinting analysis.[1] Parschat, K., et al (2007): J. Bacteriol. 189:3855-3867.[2] Ferrandez, A. et al (2000). J. Biol. Chem. 275:12214-22.[3] Garcia, B. et al (2000): Appl. Environ. Microbiol. 66:4575-8.RGP030A novel Pseudomonas putida bioreporter strain for thedetection of alkylquinolone-type quorum sensing signalmoleculesC. Müller*, S. FetznerInstitute for Molecular Microbiology and Biotechnology (IMMB),Westphalian Wilhelms-University, Münster, GermanyThe opportunistic pathogen Pseudomonas aeruginosa regulates its virulencevia a complex quorum sensing (QS) network which incorporates both N-acylhomoserine lactone and 2-alkyl-4(1H)-quinolone (AQ) signalmolecules. The >50 different AQs produced by P. aeruginosa differ mainlyin the degree of saturation and length of the alkyl chain as well as in thepresence or absence of a hydroxyl substituent at the C3-position [1]. Amongthese AQs, 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonasquinolone signal, PQS) and 2-heptyl-4(1H)-quinolone (HHQ) wereidentified as autoinducers in QS. HHQ as well as PQS act as the effectors ofthe LysR-type transcriptional regulator PqsR [2, 3].This study focuses on the validation of a lacZ-based Pseudomonas putidabioreporter strain that enables the detection of AQ signal molecules at lowconcentrations (nM to μM). P. putida KT2440 was transformed with areporter plasmid that confers constitutive expression of the pqsR gene, andcontains a transcriptional fusion of the PqsR-responsive pqsA promoter tothe reporter gene lacZ. Therefore, β-galactosidase activity is a function ofthe PqsR-stimulated transcription under the control of the pqsA promoter.The presence of HHQ or PQS (1 μM) increases the β-galactosidase activityof the bioreporter three- to four-fold compared to the activity mediated byPqsR in the absence of an effector. The bioreporter may be used to screenAQ analogues for their ability to act as HHQ/PQS agonists or antagonists,and to identify genes which encode PQS or HHQ converting enzymes.[1] Lépine, F. et al (2004): J Am Soc Mass Spectrom 15:862-869.[2] Wade, D.S. et al (2005): J Bacteriol 187:4372-4380.[3] Xiao, G. et al (2006): Mol Microbiol 62:1689-1699.spektrum | Tagungsband 2011