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

184substrate specific expression of rdh genes, the proteome of bacteriacultivated under identical growth conditions but with different electronacceptors were analysed by using a liquid chromatography tandem massspectrometric (LC-MS/MS) based approach with focus on the detected rdhproteins.A simplified sample preparation procedure was used to achieve highprotein coverage despite of low achievable cultivation densities and thesmall cell size of the bacteria. Harvested cells were lysed by mechanicaltreatment. After ultracentrifugation the membrane and cytosolic fractionwere separately digested in solution using trypsin. Desalted peptides weredirectly used for LC-MS/MS measurements. Shotgun mass spectrometryresulted in an average of 660 identified proteins which is about 44% of allpredicted gene products. Three dehalogenases were identified in allCBDB1 cultures in the presence of various halogenated hydrocarbons suchas chlorinated aromatic and non-aromatic substances. Overall 14 differentdehalogenases were detected giving first hints for different substrate-relatedexpression patterns which were determined by label-free quantification. Exactquantification of rdh proteins is planned by selected reaction monitoring (SRM)mass spectrometry. Results from these experiments will further improve theunderstanding of the correlation between the electron acceptors provided duringcultivation and expression of rdh enzymes by CBDB1.Acknowledgement: This work is supported by the DFG (research unit FOR1530).[1] Kube M. et al. (2005) Nat Biotechnol. 23: 1269-1273PSP037Identification of genes essential for anaerobic growth ofListeria monocytogenesS. Müller* 1 , A. Krementowski 1 , S. Wüstner 1 , C. Held 2 , A. Ehrenreich 2 ,S. Scherer 11 TUM, Lehrstuhl für Mikrobielle Ökologie, Freising, Germany2 TUM, Lehrstuhl für Mikrobiologie, Freising, GermanyThe adaptation of L. monocytogenes to various growth conditionscontributes to its ubiquitous distribution and its role as an important foodborne pathogen. L. monocytogenes is resistant to many adverseenvironmental conditions, e.g. it growth at temperatures from 0 - 45 °C, ina pH range from pH 4.1 to 9.6 and at high salt concentrations.Furthermore, as a facultative anaerobic bacterium it can adapt to variousoxygen tensions. Although early physiological studies on the anaerobicmetabolism of L. monocytogenes have been completed with globalapproaches such as comparative genome-, transcriptome- and proteomeanalysis, aerobic growth of L. monocytogenes is still much betterunderstood than anaerobic growth. In this study, the anaerobic growth ofL. monocytogenes was further characterized.We demonstrate that the transcriptional profile changes significantly in L.monocytogenes cells either grown aerobically or anaerobically in BHImedium at 37 °C. A set of 116 genes was stronger transcribed underaerobic conditions and a set of 26 genes was stronger transcribed underanaerobic conditions. For 19 of the 26 anaerobically stronger transcribedgenes deletion or insertion mutants were constructed. The respectivemutants are able to grow both aerobically and anaerobically, suggestingthat their expression is not essential for anaerobic growth and proliferationof L. monocytogenes.However, a high throughput screening of an insertion mutant banc (Josephet al., 2006) identified genes essential for anaerobic growth. 11 out of 1360investigated insertion mutants showed an anaerobic sensitive phenotypewhile they were able to grow aerobically. Interestingly, all these mutantsare interrupted in the atp-locus, which showed no differential transcriptiondependent on the oxygen availability. The essential function of the atplocusfor anaerobic growth was further validated by growth analysis of thedeletion mutants L. monocytogenes/atpA, L. monocytogenes/atpB andL. monocytogenes/atpD and subsequent complementation of the deletedgenes. These results indicate that the expression of a functional F 0F 1-ATPase is essential for growth and proliferation during anaerobic but notduring aerobic growth in L. monocytogenes.PSP038Biochemistry of Ethylbenzene Dehydrogenase, the key enzymeof the anaerobic ethylbenzene degradation in Azoarcus sp.strain EbN1D. Knack* 1 , A. Dudzik 2 , C. Hagel 1 , J. Heider 1 , M. Szaleniec 21 Uni Marburg, Fachbereich Biologie, Marburg, Germany2 Polish Academy of Sciences, Institute for Catalysis and SurfaceChemistry, Kraków, PolandThe initial reaction of the anaerobic degradation pathway of ethylbenzenein Azoarcus sp. strain EbN1 (“Aromatoleum aromaticum”) is an oxygenindependentand stereospecific hydroxylation of ethylbenzene to (S)-1-phenylethanol by the molybdenum/iron sulfur/heme enzyme ethylbenzenedehydrogenase (EbDH, 1). EbDH is a heterotrimer of 3 subunits with atotal molecular mass of 160 kDa and belongs to the DMSO reductasefamily of molybdenum enzymes (Type II). The subunit (96 kDa) carriesa bis-molybdopterin cofactor, which is the active site of the enzyme, and aFeS cluster. The subunit (43 kDa) carries 4 FeS clusters, which areresponsible for the electron transport from the active site of the enzyme tothehemeb cofactor in the subunit (23 kDa; 2). In the last years,comparison of kinetic data of ethylbenzene analogs which act as EbDHsubstrates as well as chromatographic analysis of the formed alcohols leadto a first model of the catalytic mechanism of the enzyme (3).Furthermore, quantum chemical calculations of the EbDH reactionmechanism were performed and supported the kinetic andchromatographic data (4). Recently, new ethylbenzene analogs were testedas possible new substrates or inhibitors of the enzyme. The kinetic data ofthese new compounds together with chromatographic data of the formedalcohol products reveal new insights into the catalytic mechanism and theenantioselectivity of the enzyme. In addition, specifically deuteratedethylbenzene derivates where synthesized to test which of the hydrogenatoms on the C1 position of the ethyl group of ethylbenzene is abstractedduring catalysis and replaced by a water-derived hydroxyl group. Kineticinvestigations and mass spectrometric analysis of the products indicate thatthe proS hydrogen is abstracted during enzymatic catalysis.1. Kniemeyer, O. Heider, J. (2001) Ethylbenzene Dehydrogenase, a Novel Hydrocarbon oxidizingMolybdenum/Iron-Sulfur/HemeEnzyme. J Biol Chem; 276:21381-213862.Kloer, D. P.; Hagel, C.; Heider, J.; Schulz, G. E. (2006) Crystal Structure of EthylbenzeneDehydrogenase from Aromatoleum aromaticum. Structure; 14:1377-13883. Szaleniec, M.; Hagel, C.; Menke, M.; Nowak, P.; Witko, M.; Heider, J. (2007) Kinetics andmechanism of oxygen-independent hydrocarbon-hydroxylation by ethylbenzene dehydrogenase.Biochem; 46:7637-76474. Szaleniec, M.; Borowski, T.; Schühle, K.; Witko, M.; Heider, J. (2010) Ab Inito Modeling ofEthylbenzene Dehydrogenase Reaction Mechanism. J Am Chem Soc; 132:6014-6024PSP039Norepinephrine and epinephrine stimulate growth andmotility of Vibrio choleraeP. Halang* 1 , T. Vorburger 1 , V. Stefanski 2 , J. Steuber 11 University of Hohenheim, Institute of Microbiology, Stuttgart, Germany2 University of Hohenheim, Institute of Animal Husbandry and Breeding,Stuttgart, GermanyUnder stress the body produces biochemical messengers likecatecholamine hormones to adapt to the specific situation. Attenuation ofthe immune response by stress hormones, together with stimulation ofbacterial growth due to hormone exposure, leads to increased susceptibilityof the host to bacterial infections.A stimulatory effect of norepinephrine (NE) and epinephrine (Epi) ongrowth ofSalmonellaserovartyphimuriumwas observed in SAPI mediumwhich contains serum proteins including the Fe-binding protein transferrin(Pullinger et al. 2010). Under these growth conditions, NE competes withtransferrin for Fe by complexation of Fe with its catechol moiety. Hereby,the availability of this important trace element increases, which in turnstimulates growth of the pathogen. NE and Epi were also shown topromote swarming ofS. typhimuriumon plates consisting of LB broth with0.3% agar (Moreira et al. 2010). We investigated the effect of NE or Epion the growth ofVibrio choleraestrain O395-N1 in SAPI-serum medium.No growth ofV. choleraestrain RIMD2203102 was observed on SAPIserummedium with added NE or Epi (Nakano et al. 2007). WithV.choleraestrain O139-N1 grown under similar conditions, NE led to a twofoldincrease in growth yield after 46 h. Both catecholamines stimulatedmotility of strain O139-N1 on SAPI-serum swarming plates, but inhibitedswarming on plates consisting of minimal medium with glucose as carbonsource. We propose that in the presence of serum proteins, the ironlimitation caused by transferrin was overcome by the catecholamines,resulting in increased motility ofV. choleraeO139-N1 compared to thehormone-free control. We suggest that in minimal medium, the ironchelatingproperties of NE and Epi led to a decrease in free iron, andresulted in diminished motility ofV. choleraecells when compared to cellsexposed to swarming plates devoid of the hormones.Moreira CG, Weinshenker D, Sperandio V (2010) QseC mediatesSalmonellaentericaserovartyphimuriumvirulence in vitro and in vivo. Infect Immun 78:914-926Nakano M et al. (2007) Catecholamine-induced stimulation of growth in Vibrio species. Lett ApplMicrobiol 44:649-653Pullinger GD et al. (2010) Norepinephrine augmentsSalmonella enterica-induced enteritis in amanner associated with increased net replication but independent of the putative adrenergic sensorkinases QseC and QseE. Infect Immun 78:372-380PSP040Three different principles of ketone carboxylation inAromatoleum aromaticumK. Schuehle*, D. Kleinsorge, J. HeiderPhilipps-Universität Marburg, Mikrobiologie, Marburg, GermanyThe b-proteobacterium Aromatoleum aromaticum degrades aliphatic andaromatic ketones (e.g. acetone, butanone, acetophenone, 4-hydroxyacetophenone) as single substrates under aerobic and denitrifyingconditions. Degradation of each substrate is initiated by a carboxylationreaction catalysed by specific, substrate-induced carboxylases.Acetone carboxylase (Acx) carboxylates acetone and butanone in an ATPdependent,biotin-independent reaction. 2 ATP are hydrolyzed to 2 AMPand 4 Pi for one acetone carboxylated. Acx is present in cells grownaerobically or anaerobically on acetone or butanone, but not in cells grownBIOspektrum | Tagungsband 2012