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

196down of RSs2430 influences the expression of photosynthesis genes inRhodobacter sphaeroides.Northern blots showed that RSs2430 is processed, whereby different3’ends are generated. The different 3’ends were identified by 3’RACE.Interestingly, only the processed RSs2430-fragments, not the primarytranscript, were enriched in the overexpression strain. By using real timeRT-PCR and microarray analyses we showed that overexpression ofRSs2430 results in a decreased expression of photosynthesis genes.To study the interaction of RSs2430 and its target mRNAs, a lacZ based invivo reporter system was used. We observed specific translation repressionof a light-independent protochlorophyllide reductase subunit N (bchN)under high and low oxygen growth conditions.1. Berghoff, B.A., Glaeser, J., Sharma, C.M., Vogel, J. and Klug, G. (2009) Photooxidative stressinducedand abundant small RNAs in Rhodobacter sphaeroides. Mol. Microbiol.,74(6), 1497-512.RSP006Examination of a timing mechanism in Rhodobacter sphaeroidesY. Hermanns* 1 , N. Schürgers 2 , K. Haberzettl 1 , A. Wilde 2 , G. Klug 11 Institut f. Mikro- und Molekularbiologie, Klug, Giessen, Germany2 Institut f. Mikro- und Molekularbiologie, Wilde, Giessen, GermanyTiming mechanisms are known for over 250 years in eukaryotes. Untilnow amongst prokaryotes only cyanobacteria could be shown to possess asystem to measure time. In Synechococcus elongatus a circadian clockbuilds upon an oscillator of three proteins, KaiA, KaiB and KaiC. Aphosphorylation of KaiC in a circadian manner could be shown in vitro[1]. All three proteins are essential for clock function. Accordingly, mostcyanobacteria possess at least one copy of each gene. An exception is themarine cyanobacterium Prochlorococcus marinus, which has suffered astepwise deletion of the kaiA gene [2] but retains a 24 hour rhythm inDNA replication, which is strongly synchronized by alternation of day andnight cycles. Surprisingly, the facultative phototrophic proteobacteriumRhodobacter sphaeroides possesses a cluster of kaiBC genes similar toProchlorococcus. Therefore it has been hypothesized that R. sphaeroidesmay exhibit a rhythmic behavior in gene expression. Such a rhythm hasbeen reported earlier via a luciferase reporter gene system [3]. Bymicroarray analysis, we were able to show a decrease in the expression ofphotosynthesis genes in a continuously growing R. sphaeroides cultureafter 12 hours of illumination with white light. Preveniently this culturehad been put under a 12 hour light/dark rhythm for two days. This datasuggests an adaptation to a returning environmental cycle and theexistence of a functional timing mechanism in purple photosyntheticbacteria. Furthermore, by an in vitro phosphorylation assay an autokinaseactivity for RspKaiC could be shown which is not altered by the presenceof RspKaiB. Future results may shed some light on the existence andevolution of clock systems and circadian rhythms in prokaryotes other thancyanobacteria.[1] M. Nakajima, Science.(2005),308, 414-415. [2] J. Holtzendorff, Journal of BiologicalRhythms(2008),23, 187-199. [3] H. Min, FEBS letters.(2005),579808-812.RSP007Role of the Irr protein in the regulation of iron metabolism inRhodobacter sphaeroidesB. Remes*, V. Peuser, G. KlugInstitut für Mikro- und Molekularbiologie, AG Klug, Gießen, GermanyIron is an essential element for all living organisms. However, since ironpotentiates oxygen toxicity by the production of hydroxyl radicals in theFenton reaction, life in the presence of oxygen requires a strict regulationof iron metabolism.The Fur family of proteins are well analyzed proteins that regulatetranscription of genes in response to iron availability in bacteria (1). Inalpha-proteobacteria little is known about the iron mediated generegulation. The available experimental data suggest that iron regulationmainly occurs by regulators different from Fur (2). The Irr (iron responseregulator) protein and its orthologues form a distinct sub-branch of the Fursuperfamily but occur only in members of the Rhizobiales andRhodobacterales and few other genera. Most iron-dependent genes inalpha-proteobacteria are regulated positively or negatively by Irr (3). Athigh iron concentration Irr is degraded. ROS seem to promote thisdegradation indicating another link between iron metabolism and oxidativestress (4). We studied the role of the Irr homologue RSP_3179 in thephotosynthetic alpha-proteobacterium Rhodobacter sphaeroides.While Irr had little effect on growth under iron-limiting or non-limitingconditions its deletion resulted in increased resistance to hydrogenperoxide and singlet oxygen. This correlates with an elevated expression ofkatE for catalase in the Irr mutant compared to the wild type under nonstressconditions. Transcriptome studies revealed that Irr strongly affectsthe expression of genes for iron metabolism, but also has some influenceon genes involved in stress responses, citric acid cycle, oxidativephosphorylation, transport, and photosynthesis. Most genes showed higherexpression levels in the wild type than in the mutant under normal growthconditions indicating an activator function of Irr. Irr was however notrequired to activate genes of the iron metabolism in response to ironlimitation. This was also true for genes mbfA and ccpA, which wereverified as direct targets of Irr.1. Hantke, K. (2001) Iron and metal regulation in bacteria. Curr Opin Microbiol 4: 172-177.2. Johnston, A.W., Todd, J.D., Curson, A.R., Lei, S., Nikolaidou-Katsaridou, N., Gelfand, M.S., andRodionov, D.A. (2007) Living without Fur: the subtlety and complexity of iron-responsive gene regulationin the symbiotic bacterium Rhizobium and other alpha-proteobacteria. Biometals 20: 501-511.3. Rudolph G, Semini G, Hauser F, Lindemann A, Friberg M, et al. (2006) The Iron control element, actingin positive and negative control of iron-regulated Bradyrhizobium japonicum genes, is a target for the Irrprotein. J Bacteriol 188: 733-744.4. Yang, J., Panek, H.R., and O'Brian, M.R. (2006a) Oxidative stress promotes degradation of the Irr proteinto regulate haem biosynthesis in Bradyrhizobium japonicum. Mol Microbiol 60: 209-218.RSP008Anaerobic toluene metabolism: First evidences for twosubtypes of benzylsuccinate synthaseS. Kümmel* 1 , K. Kuntze 2 , C. Vogt 1 , M. Boll 2 , H.-H. Richnow 11 Helmholtz Centre for environmental research - UfZ, Isotope Biogeochemistry,Leipzig, Germany2 Universität Leipzig, Institut für Biochemie, Leipzig, GermanyThe aromatic hydrocarbon toluene can be degraded in the absence ofoxygen by various facultative or obligate anaerobic bacteria using nitrate,sulphate or Fe(III) as terminal electron acceptor. In all tested strains so far,toluene is activated by an addition reaction of the toluene methyl group tothe double bond of fumarate to form benzylsuccinate. This reaction iscatalyzed by the benzylsuccinate synthase (Bss), which is a member of theglycyl radical family of enzymes. Even if the overall reaction, catalysed bythe Bss, is in all tested strains the same, the alignment of available Bssgene sequences reveals that they slightly differ. This result leads to apossibility to distinguish between several Bss isoenzymes on a geneticlevel.In previous in vivo studies, data from two dimensional compound specificstable isotope analyses (2D-CSIA) indicated that the reaction mechanismof Bss subtypes in facultative and obligate anaerobes may differ. To testwhether the observed isotope fractionation effects are directly due to theBss reaction mechanisms, in vitro assays were performed using cell-freeextracts of different facultative and obligate anaerobic toluene degraders.In addition, the enzymatically mediated exchange of hydrogen atomsbetween toluene and the solvent was investigated. The results of bothapproaches confirmed the hypothesis that at least two mechanisticallydifferent subtypes of Bss exist: one occurring in facultative anaerobes andone occurring in obligate anaerobes. Thus, 2D-CSIA may allowspecifically detecting toluene degradation by facultative or obligateanaerobes at contaminated field sites.RSP009Characterization of AHL-lactonases and their influence on thequorum sensing system of Vibrio harveyiM. Reiger*, C. Anetzberger, K. JungBiozentrum LMU München, Biologie I, Mikrobiologie, Planegg-Martinsried, GermanyBacteria use signaling molecules, so called autoinducers (AIs) tocommunicate and to monitor their environment. The marine -proteobacterium Vibrio harveyi uses three different classes of AIs forcommunication, HAI-1, a N-(3-hydroxybutyryl)-D-homoserine lactone(AHL), AI-2, a furanosylborate diester and CAI-1, a (Z)-3-aminoundec-2-en-4-on. Thereby type III secretion, siderophore production, exoproteolyticactivity, biofilm formation, and bioluminescence are regulated.Heterogeneous behavior of the wild type population with respect tobioluminescence was shown before (Anetzberger et al., 2009). Theaddition of an excess of exogenous AIs resulted in a homogeneouspopulation. It is suggested that the population is able to tightly control theextracellular AI concentrations. V. harveyi has five genes encodingputative lactonases.The putative lactonase VIBHAR_02708, which is highly conserved amongVibrio species, was purified and characterized. UPLC coupled MS analysisof the HAI-1 cleavage products confirmed that VIBHAR_02708 is a lactonase.Subsequently, the corresponding deletion mutant was constructed andcharacterized. The HAI-1 concentration in the culture fluid was about 30%higher in the VIBHAR_02708 mutant than in the wild type. These dataclearly show an influence of the lactonase VIBHAR_02708 on the QS systemof V. harveyi via the adjustment of the HAI-1 concentration.Anetzberger, C., Pirch, T. and Jung, K. (2009), Heterogeneity in quorum sensing-regulatedbioluminescence of Vibrio harveyi. Molecular Microbiology, 73: 267-277.RSP010Targeted proteome analysis of Corynebacterium glutamicumR. Voges*, B. Klein, M. Oldiges, W. Wiechert, S. NoackFZ Juelich, IBG1:Biotechnology, Juelich, GermanyThe Gram positive soil bacterium C. glutamicum is a widely used hostorganism in industrial biotechnology [1]. Main products are the aminoacids L-glutamate, L-lysine and L-threonine. New desired products includebuilding blocks for chemical industry, biofuels and heterologous proteins.BIOspektrum | Tagungsband 2012