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

162Streptomyces sp. strain FLA shows the highest activity with Ni 2+ ascofactor, followed by Co 2+ [2, 3] . The presumed ligand residues H69, H71and H115 of QueD were individually replaced by alanine. Whereas QueD-H69A and QueD-H115A exhibited almost the same metal occupancy asthe wild type protein (about 0.8 equivalents of nickel per proteinmonomer), QueD-H71A contained about 0.4 equivalents of nickel permonomer, indicating that H71 is important for metal binding. Replacementof H115 had only minor effects on the activity of the enzyme, whereassubstitution of H71 or H69 resulted in enzymatic inactivation.Interestingly, anoxic fluorescence titration experiments indicated that theQueD-H69A protein is still able to bind quercetin with a K d similar to thatof the wild-type enzyme, suggesting that the H69 residue is relevant incatalysis rather than substrate binding. Substrate deprotonation has beendiscussed as the initial reaction step catalysed by quercetinases [4] . TheH69 residue may act as the general base catalyst for initial deprotonationof the metal-bound quercetin.Preference for Ni 2+ is extraordinary for oxygenases, raising the question ofwhether the metal ion has a redox role in catalysis. The quercetinasereaction has been proposed to involve single electron transfer from theflavonolate anion via the metal to dioxygen [4, 5] . However, Ni 2+ centers inligand environments dominated by O- and N-donors were proposed to beredox inert [6] . Construction and characterisation of a Zn 2+ isoform ofQueD ion could shed light upon the question of whether the metal acts asan electron conduit. Since in vivo incorporation of Zn 2+ into cytosolicrecombinant QueD failed, periplasmic expression and in vitrotranscription/translation studies are currently being performed.[1]Dunwell JM, Culham A, Carter CE, Sosa-Aguirre CR, Goodenough PW (2001) Trends Biochem. Sci.26:740-746[2]Merkens H, Sielker S, Rose K, Fetzner S (2007) Arch. Microbiol. 187:475-487[3]Merkens H, Kappl R, Jakob RP, Schmid FX, Fetzner S (2008) Biochemistry 47:12185-12196[4]Steiner RA, Kalk KH, Dijkstra BW (2002) Proc. Natl. Acad. Sci. USA 99:16625-16630[5]Schaab MR, Barney BM, Francisco WA (2006) Biochemistry 45:1009-1016[6]Maroney MJ (1999) Curr. Opin. Chem. Biol. 3:188-199OTP113Unravelling the role of small non-coding RNAsinMethanosarcina mazeiGö1D. Prasse* 1 , D. Jäger 1 , S. Pernitzsch 1,2 , A. Richter 3 , R. Backofen 3 , C. Sharma 2 ,R.A. Schmitz-Streit 11 Department of General Microbiology , Christian-Albrechts-University, Kiel,Germany2 Institute for Molecular Infection Biology, Julius-Maximilians-University,Würzburg, Germany3 Department of Computer Science , Albert-Ludwigs-University, Freiburg,GermanyIn recent years the global impact of small non-coding RNAs (sRNA) in alldomains of life comes more and more obvious. As still little is known onregulatory roles of sRNAs in the domain of Archaea, we recentlyperformed a genome-wide RNA-seq approach, resulting in the discoveryof 248 sRNAs in Methanosarcina mazeistrain Gö1 [1]. The archaeal modelorganism M. mazeiis a representative methylotrophic archaeon ofsignificant ecological importance due to its role in biogenic methaneproduction in various anaerobic habitats on Earth and is able to fixmolecular nitrogen. Here we present the characterization of one selectedsRNA, sRNA 162, using biochemical and genetic approaches. Therespective results will be discussed in order to elucidate the potentialregulatory role of sRNA 162 inM. mazei.1. Jäger D , Sharma CM , Thomsen J, Ehlers C, Vogel J, Schmitz RA (2009) Deep sequencinganalysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability.PNAS. 106(51):21878-21882OTP115Changes in the microbial community structure of a fjord as aresult of ecologically engineered oxygenation (Byfjorden, westernSweden)M. Forth* 1 , B. Liljebladth 2 , A. Stigebrandt 2 , P. Hall 3 , A. Treusch 11 University of Southern Denmark, Institute of Biology, Odense C, Denmark2 University of Gothenburg, Department of Earth Sciences, Gothenburg, Sweden3 University of Gothenburg , Marine Chemistry, Gothenburg, SwedenThe availability of oxygen has a high influence on the diversity ofcommunities and the distribution of organisms in pelagic ecosystems.Hypoxic or anoxic conditions caused e.g. by stratification lead to reducedhabitats for oxygen depending eukaryotic and prokaryotic life. In recentyears, oxygen depleted bodies of water are becoming more common. It isexpected that in the near future anthropogenic influences like e.g. climatechange and agriculture will intensify this problem. Recently, more efforthas been put into the restoration of hypoxic habitats. TheBaltic deep-waterOXygenation(BOX) project proposed to introduce oxygen into the longtermhypoxic or anoxic bottom waters of the Baltic Sea by using winddriven pumps to generate artificial mixing.The Swedish Byfjorden is a long-term stratified system with a lower watercolumn and benthic zone that has been anoxic for a long time. In addition,an inflow of freshwater from a river is generating a brackish, welloxygenatedlayer of surface water with lower salinity than the deeperlayers, strengthening the stratification. Because of this, the Byfjorden is anideal model system for the Baltic Sea. As a part of the BOX project, a pilotstudy to test the artificial oxygenation was started in 2009. A pump wasinstalled in the Byfjorden to mix the surface water into the deeper layersand thereby oxygenate the anoxic zone.In this study, we monitored changes in microbial community structure inresponse to the oxygenation project in the Byfjorden. We analyzed watercolumn samples from before and during the oxygenation as well as from acontrol station in a nearby, natural oxic fjord using a molecular microbialcommunity profiling method. Here, we present the results in the context ofbiogeochemical and hydrographical data to show the impact of theoxygenation on the bacterial and archaeal community structures.OTP116Gene cluster for biosynthesis of the catechol-peptidesiderophore griseobactin in Streptomyces griseusS.I. Patzer*, V. BraunMax Planck Institute for Developmental Biology, Tübingen, GermanyIron is an essential element for the growth and proliferation of nearly allmicroorganisms. In the presence of oxygen, soluble ferrous iron is readilyoxidized to its ferric form, which is predominantly insoluble at neutral pH.To overcome iron limitation, many bacteria synthesize and secrete lowmolecular-weight,high-affinity ferric iron chelators, called siderophores,which are actively taken up as a complex with Fe 3+ by a cognate ABCtransport system. The main siderophores produced by streptomycetes aredesferrioxamines.Here we show that several Streptomyces griseus strains, in addition,synthesize a hitherto unknown siderophore with a catechol-peptidestructure, which we named griseobactin. The production is repressed byiron. We sequenced a 26-kb DNA region comprising a siderophorebiosynthetic gene cluster encoding proteins similar to DhbABCEFG,which are involved in the biosynthesis of 2,3-dihydroxybenzoate (DHBA)and in the incorporation of DHBA into siderophores via a nonribosomalpeptide synthetase. Adjacent to the biosynthesis genes are genes thatencode proteins for the secretion, uptake, and degradation of siderophores.Knockout mutagenesis, complementation and heterologous expressionconfirmed the requirement of the dhb genes for synthesis and secretion ofDHBA and of the entire biosynthesis gene cluster for biosynthesis andsecretion of griseobactin. Griseobactin was purified and characterized; itsstructure is consistent with a cyclic and, to a lesser extent, linear form ofthe trimeric ester of 2,3-dihydroxybenzoyl-arginyl-threonine complexedwith aluminum under iron-limiting conditions. This is the first report onthe identification of the genes responsible for DHBA and catecholsiderophore biosynthesis in Streptomyces.Patzer S. I., Braun V. (2010) J. Bacteriol. 192:426-35OTP117Biochemical and genetic characterization of ethylene glycolmetabolism in Pseudomonas putida KT2440 and JM37B. Mückschel* 1 , O. Simon 2 , J. Klebensberger 1 , N. Graf 3 , J. Altenbuchner 3 ,J. Pfannstiel 2 , A. Huber 2 , B. Hauer 11 Universität Stuttgart, Institute of Technical Biochemistry, Stuttgart, Germany2 Universität Hohenheim, Department of Biosensorics, Stuttgart, Germany3 Universität Stuttgart, Institute of Industrial Genetics, Stuttgart, GermanyBeing an important building block for flavor chemicals and polymers,glyoxylic acid is a valuable product for many industrial processes. Theenzymatic oxidation of ethylene glycol could provide an interestingalternative to the commonly used chemical synthesis of glyoxylic acid. Inorder to develop such a biocatalyst, we started to investigate themetabolism of ethylene glycol using the Pseudomonas putida strainsKT2440 and JM37.We found that P. putida JM37 rapidly grows in minimal media containingethylene glycol or glyoxylic acid as sole source of carbon and energy,while strain KT2440 did not show growth even after three days ofincubation. However, experiments with dense cell suspensions revealedcomplete conversion of ethylene glycol for both strains. In contrast toJM37, strain KT2440 showed temporal accumulation of glycolic acid andglyoxylic acid as intermediates, finally yielding oxalic acid as the endproduct.To identify key enzymes involved in the metabolism of ethylene glycol, adifferential proteomic approach was used. Increased expression oftartronate semialdehyde synthase (Gcl), malate synthase (GlcB), andisocitrate lyase (AceA) in strain JM37 as well as AceA in strain KT2440was found during incubations with ethylene glycol or glyoxylic acid. Acorresponding triple mutant strain harboring an additional deletion inprpB, encoding for methyl isocitrate lyase, was constructed andcharacterized in strain KT2440. This mutant showed a significantreduction in the conversion of ethylene glycol and increased accumulationof glycolic acid and glyoxylic acid compared to the wildtype strain.Further analysis uncovered the induction of two PQQ-dependant ethanolBIOspektrum | Tagungsband 2012