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

187PSP049Characterization of plasmid pPO1 from the hyperacidophilePicrophilus oshimaeA. Angelov* 1 , J. Voss 2 , W. Liebl 11 Technische Universität München, Lehrstuhl für Mikrobiologie, Freising,Germany2 Georg-August-Universität Göttingen, Institut für Mikrobiologie und Genetik,Göttingen, GermanyPicrophilus oshimae and Picrophilus torridus are free-living, moderatelythermophilic and acidophilic organisms from the lineage ofEuryarchaeota. With a pH optimum of growth at pH 0.7 and the ability toeven withstand molar concentrations of sulphuric acid, these organismsrepresent the most extreme acidophiles known. So far, nothing is knownabout plasmid biology in these hyperacidophiles. Also, there are no genetictools available for this genus. We have mobilized the 7.6 Kbp plasmidfrom P. oshimae in E. coli by introducing origin-containing transposonsand describe the plasmid in terms of its nucleotide sequence, copy numberin the native host, mode of replication and transcriptional start sites of theencoded ORFs. Plasmid pPO1 may encode a restriction/modificationsystem in addition to its replication functions. The information gainedfrom the pPO1 plasmid may prove useful in developing a cloning systemfor this group of extreme acidophiles.PSP050The three NiFe-hydrogenases of Sulfurospirillum multivorans:Insights into the hydrogen metabolism of an organohaliderespiring bacteriumX. Wei* 1 , C. Schiffmann 2 , J. Seifert 2 , T. Goris 1 , G. Diekert 11 Friedrich Schiller University, Department of Applied and EcologicalMicrobiology, Jena, Germany2 Helmholtz-Centre for Environmental Research - UFZ, DepartmentProteomics, Leipzig, GermanyOne of the simplest reactions in nature, the oxidation of molecularhydrogen and its reverse reaction, is catalysed by a group of enzymescalled hydrogenases. Opposed to the simplicity of this reaction,hydrogenases are complex enzymes with several metal-containingcofactors. They appear in multifaceted forms, often in one single organism,where they fulfill different physiological roles. One of the largest groupsof hydrogenases harbour one nickel and one iron atom in their catalyticcenter. Thus, they are called NiFe-hydrogenases.Sulfurospirillum multivorans, an organohalide-respiring -proteobacterium, harbours the genes coding for at least three NiFehydrogenases,none of them hitherto investigated. The most prominent roleof energy conservation via the oxidation of H 2 is fulfilled presumably by amembrane-bound uptake hydrogenase, similar to the MBH of Wolinellasuccinogenes. The same gene cluster comprises a second hydrogenase,whose physiological role is unclear. It is similar to hydrogenase 3 fromAquifex aeolicus and, to a lesser extent to regulatory hydrogenases andcyanobacterial uptake hydrogenases. The third hydrogenase, encoded byfour genes spatially separated from the other hydrogenase gene cluster, isrelated to H 2-evolving energy converting hydrogenases (Ech) and mightact as an electron sink, as we have detected H 2 production duringmicroaerobic growth after depletion of oxygen. Remarkably, themembrane subunits normally present in these hydrogenases, referring to aproton pump and an electron-transferring subunit of complex I, are missingon the according S. multivorans gene cluster. This raises the question,whether the enzyme binds to the according proteins of the respiratorychain present in the organism, or if it resides freely in the cytoplasm. Inorder to understand the physiological role of these so far undercharacterisedNiFe-hydrogenases in S. multivorans, growth experiments,transcription analysis and subcellular localisation studies accompanied byactivity measurements were carried out, whereas purification,spectroscopical analyses and genetic modifications are planned.Acknowledgement: This work is supported by the DFG (research unit FOR1530)PSP051Analysis of the dual flagellar stator system in Shewanellaoneidensis MR-1 at the single-cell levelA. Paulick*, K. ThormannMax-Planck-Institute for Terrestrial Microbiology, Ecophysiology,Marburg, GermanyFlagella are rotating filaments driven by a motor complex at the filamentsbase which is powered by the sodium- or proton-motive force. The motorconsists of two major structures, the rotating switch complex and the statorcomplexes that surround the rotor in a ring-like fashion. The statorcomplexes within this stator ring system are constantly exchanged with amembrane-located pool of precomplexes that are activated uponincorporation into the motor.Recent studies on the gammproteobacterium Shewanella oneidensis MR-1revealed that two different sets of stators, annotated as PomAB andMotAB, differentially support the rotation of a single polar flagellum.PomAB, the dominant stator complex, is sodium-ion dependent, andMotAB, most likely acquired by lateral gene transfer, is proton dependent.Physiological and localisation studies provide evidence that the rotor-statorconfiguration in the flagellar motor is adjusted to environmental sodiumionconcentrations through an exchange of stator complexes. Both statorsappear to be simultaneously incorporated into the flagellar motor underlow sodium-ion concentrations, suggesting that S. oneidensis MR-1 has ahybrid motor that concurrently uses sodium-ions and protons. A globaldatabase analysis of bacterial genomes revealed that dual or multiple statorsystems are surprisingly common among bacteria. To demonstrate, for thefirst time, the existence of a naturally occuring flagellar hybrid motor,flagellar performance was analysed at the single cell level. To this end, ‘tetheredcell’ and ‘bead’-assays were established. Using these assays in concert withfluorescent microscopy on labeled stator components, we performed in vivoanalysis of the stator ring composition and dynamics. The results give insightsinto the dynamic adaption of the flagellar motor configuration in dependence ofthe environmental sodium-ion concentrations.PSP052Activity and localization of Dehydrogenases in GluconobacteroxydansS. Kokoschka*, S. Lasota, M. Enseleit, M. HoppertUniversität Göttingen, Institut f. Mikrobiologie und Genetik, Göttingen,GermanyBacterial cytoplasmic and intracytoplasmic membranes are importantmounting plates for all types of proteins directly or indirectly involved inelectron transport and generation of proton gradients. In Gluconobacteroxydans membrane-bound dehydrogenases are exposed to the periplasmand funnel reducing equivalents from educts to the electron transportchain, thereby releasing diverse incompletely oxidized products. Here, weanalyze the activities and expression of membrane-bound dehydrogenasesof Gluconobacter oxydans under different growth conditions with ethanol,glucose, glycerol, mannitol and sorbitol as substrates. Osmotic stress andoxygen partial pressure increases specific activities by up to one order ofmagnitude. Measurements of enzyme activities were also supported byimmunolocalization of two key enzymes, the PQQ-dependant membraneboundsorbitol dehydrogenase and the quinol oxidase in Gluconobactercells. This technique allows a semi-quantitative estimation of enzymeexpression and, at the same time, localization at subcellular level.PSP053Alternative fructose utilization in CorynebacteriumglutamicumS.N. Lindner* 1 , I. Krahn 1 , D. Stoppel 2 , J.P. Krause 1 , V.F. Wendisch 11 University of Bielefeld, Genetics of Prokaryotes, Bielefeld, Germany2 Westfalian Wilhelms University Münster, Münster, GermanyCorynebacterium glutamicum is used for the industrial scale production ofamino acids, such as the feed additive L-lysine or the flavor enhancer L-glutamate. Predominantly the fermentation of amino acids is carried outusing sugar substrates, such as glucose, sucrose, and fructose, which are allsubstrates of the phosphotransferase system (PTS) in C. glutamicum. Theutilization of fructose starts by PTS mediated uptake and simultaneousphosphoenolpyruvate dependent phosphorylation to fructose-1-phosphate.Subsequently fructose-1-phosphate is phosphorylated to the glycolyticintermediate fructose-1,6-bisphosphate by 1-phosphofructokinases.To analyze the role of the 1-phosphofructokinases in C. glutamicumdeletion mutants of the corresponding genes fruK1 and/or fruK2 wereconstructed. The presence of one of the 1-phosphofructokinase genes wassufficient for growth with fructose whereas fruK1 encoded the moreimportant 1-phosphofructokinase as only fruK1 and not fruK2 showedimpaired growth compared to the WT. Growth with fructose wascompletely inhibited when both genes fruK1 and fruK2 were deleted(fruK1fruK2).Suppressor mutants were isolated after prolonged incubation offruK1fruK2 in fructose minimal medium. These suppressor mutantsregained the ability to grow from fructose. Growth rates of the suppressormutants were comparable to the WT with a concomittant increase ofbiomass yields of the suppressor mutants. The biomass increase is likelydue to the reduced acid byproduct formation. When testing for L-lysineproduction from fructose, the suppressor mutants showed strong increasedL-lysine production compared to the parental strain.BIOspektrum | Tagungsband 2012