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

148using real-time PCR. Activity measurements and analysis of spatialrelationship are planned via fluorescencein situhybridization (FISH).The molecular fingerprinting revealed an altered microbial biocenosisduring a foam formation event and over a one-year period in the foamingpronereactor. Microthrix parvicella and Cloacamonas acidaminovoransseemed to be directly connected to the foam formation. Higher cellnumbers of these two organisms were detected in the foam. Real-time PCRmeasurements verified higher DNA amounts of M. parvicella in thefoaming reactor and foam. Additionally, higher cell numbers of M.parvicella could be detected in the winter months possibly caused due totemperature sensitivity.M. parvicella and C. acidaminovorans could act as indicator organisms fora starting foam formation in large-scale biogas plants. Finding a thresholdDNA concentration of M. parvicella or C. acidaminovorans could serve asearly-warning indicator to take countermeasures against a foam formation.OTP047Monomerization of the dimeric polyprenylglyceryl phosphatesynthase PcrB by protein design results in a different substratespecificityD. Peterhoff*, H. Zellner, R. Merkl, R. Sterner, P. BabingerUniversity of Regensburg, Biophysics and physical Biochemistry,Regensburg, GermanyThe bacterial PcrB proteins show about 35% sequence identity to thearchaeal geranylgeranylglyceryl phosphate synthases (GGGPS). PcrB hasrecently been shown to be a heptaprenylglyceryl phosphate synthase,which catalyzes the formation of an ether bond between sn-glycerol-1-phosphate (G1P) and heptaprenyl pyrophosphate (HepPP) [1-2] . The crystalstructure of Bacillus subtilis PcrB reveals a G1P-binding site as well as along hydrophobic groove similar to the geranylgeranyl pyrophosphatebinding site of Archaeoglobus fulgidus GGGPS [3-4] . However, the “ruler”limiting the length of the polyprenyl pyrophosphate to 20 C-atoms inGGGPS is missing in PcrB, allowing the binding of HepPP which contains35 C-atoms.Both GGGPS and PcrB form homodimers. The subunit interface has beenunambiguously determined for GGGPS, whereas the published contactbetween the two PcrB subunits [3] is implausible due to the relatively smallburied surface area. We therefore decided to identify the native contactinterface of PcrB and to study the impact of dimerization for proteinstability and substrate specificity. Bioinformatic analysis predicted twoalternative interfaces, one of them being identical to the GGGPS interface.In order to loosen the dimer, we introduced destabilizing amino acidsindividually into the two predicted interfaces. Monomerization wasexclusively observed with mutations in the surface area that corresponds tothe GGGPS interface. Furthermore, we incorporated the non-naturalaminoacid p-azido-L-phenylalanine at specific sites into each potentialinterface using the method developed by Schultz and coworkers [5] tocrosslink the protomers. The experiment confirmed that PcrB has the samecontact interface like GGGPS. The stability of the monomerized variantswas not severely affected. However, their substrate specificity was limitedto shorter polyprenyl pyrophosphates (geranyl pyrophosphate, 10 C-atoms). This finding shows that dimerization of PcrB is a prerequisite tobind and process the native polyprenyl pyrophosphate substrate.[1] H. Guldan, R. Sterner, P. Babinger, Biochemistry 2008, 47, 7376-7384.[2] H. Guldan, F. M. Matysik, M. Bocola, R. Sterner, P. Babinger, Angewandte Chemie Int. Ed. 2011, 50,8188-8191.[3] J. Badger, J. M. Sauder, J. M. Adams, S. Antonysamy, K. Bain, M. G. Bergseid, S. G. Buchanan, M. D.Buchanan, Y. Batiyenko, J. A. Christopher, et al., Proteins 2005, 60, 787-796.[4] J. Payandeh, E. F. Pai, J Mol Evol 2007, 64, 364-374.[5] T. S. Young, I. Ahmad, J. A. Yin, P. G. Schultz, J Mol Biol 2010, 395, 361-374.OTP048Phylogenetic relationships among bacteria described fromalgae: Distinct source of new taxaF. Goecke, V. Thiel, J. Wiese*, A. Labes, J.F. ImhoffGEOMAR | Helmholtz-Zentrum für Ozeanforschung Kiel, Kieler Wirkstoff-Zentrum am GEOMAR, Kiel, GermanyBacteria are an inherent part of the physical environment of algae. Algaeare key components of the aquatic environments and are substrates formillions of microorganisms waiting to be discovered. Recentinvestigations have shown that bacterial communities associated withalgae are highly specific to their host. Worldwide, representatives ofseveral new bacterial species and genera have been isolated from algae.We conducted a phylogenetic study based on 16S rRNA gene sequencesavailable in GenBank of 101 bacterial species (only type strains) whichhave been described as new species and have been derived from eukaryoticmacro- and micro-algal sources. We found a clear dominance of 6 majorbacterial lineages. The major lineage corresponded to Bacteroidetes with42 newly described bacterial species, followed by Proteobacteria(including Alpha- and Gammaproteobacteria) with 36 species. Firmicutes,Actinobacteria, Verrucomicrobia and Planctomycetes contributed to alesser extent. Based on the information of the species descriptions, 32% ofall new bacterial species were able to decompose macroalgalpolysaccharides, especially the members of Bacteroidetes andGammaproteobacteria. On the other hand, most of the bacteria describedfrom marine microalgae grouped into the Alphaproteobacteria, indicatingthat some members of this group are well adapted to live in closeassociation with phytoplankton. We confirmed algae as a distinct sourcefor new bacterial taxa. Although such associations can be random orspecific, they could be explained by evolutionary adaptations throughmetabolic pathways, niche specificity or mutualistic relationships. Thoseparameters might play an important role in algae-bacteria relationships innature.OTP049Novel Octaheme Cytochromes c enzymesB. Hermann* 1 , F. Kemper 1 , M. Braun 1 , S. Netzer 1 , M. Dietrich 1 , M. Kern 2 ,J. Simon 2 , D. Wohlwend 1 , O. Einsle 11 Albert-Ludwigs-Universität Freiburg, Institut for Organische Chemie undBiochemie, Freiburg, Germany2 TU Darmstadt, Biologie, Darmstadt, GermanyMultiheme Cytochromes c (MCC) are a diverse family of electron carriersand redox enzymes that play a central role in several metabolic pathways.Some MCC enzymes have been structurally characterized in the past andwere found to contain conserved heme-packing motifs, although theirprimary structures are largely unrelated [1,2]. Interestingly, purified MCCsare able to convert more than one substrate. However these activities haveto be interpreted carefully for the fact that not every measured in vitroactivity has a compulsory physiological role.The classical enzyme displaying a wide substrate versatility is NrfA, anammonium-producing pentaheme cytochrome c nitrite reductase, thatcatalyses the six-electron reduction of nitrite to ammonia as the keyreaction in respiratory nitrite ammonification. It is also able to converthydroxylamine, nitric oxide, and sulfite [3,4]. Other already characterizedMCCs belong to the family of Octaheme Cytochomes C (OCC), likeoctaheme cytochrome c nitrite reductase (Onr) [5], octaheme tetrathionatereductase (Otr)[6] or the hydroxylamine oxidoreductase (HAO) [7]. Thelatter is so far the only OCC known to function as an oxidase. This ismainly due to an unusual cross-link of a tyrosine with a heme meso carbonof the active-site heme.Another so far uncharacterized class of OCC are the HAO, found in someEpsilonproteobacteria, such as some Campylobacter species [8]. Theseorganisms lack a NrfA homologue and yet are reported as nitriteammonifiers. Although the enzymes clearly are related to ’classical’ HAO,the active-site tyrosine residue is absent in HAO. It has been hypothesizedthat this enzyme reduces nitrite to hydroxylamine but it might just as wellperform nitrite reduction to ammonium, thereby functionally replacingNrfA.To broaden our knowledge of MCCs we focus on the structural propertiesthat lead to substrate versatility of MCCs. Therefore we use highresolutionX-ray crystallography in combination with in vitro activityassays.As a first step we were able to purify two octaheme HAO, fromCampylobacter curvus and Campylobacter concisus and observed nitritereductase activity which is indeed lower than NrfA activity but still highenough to play a physiological role.[1] Einsle O. et al., Nature, 1999, 400, 476-480[2] Mowat, C.G. et al., Dalton Trans, 2005, 7, 3381-3389[3] Rudolf, M. et al., Biochem. Soc. Trans, 2002, 30, 649-653[4] Lukat, P. et al., Biochemistry, 2008, 47, 2080-2086[5] Tikhonova, T.V. et al., BBA, 2006, 1764, 715-723[6] Mowat, C.G. et al., Nat. Struct. Mol. Biol., 2004, 11, 1023-1024[7] Igarashi, N. et al., Nat. Struct. Biol., 1997, 4, 276-284[8] Kern, M. et al., BBA, 2009, 1787, 646-656OTP050Characterization of the potential heme chaperone HemWV. Haskamp*, S. Huhn, M. Jahn, D. JahnTU Braunschweig, Institut für Mikrobilogie, Braunschweig, GermanyModified tetrapyrroles are complex macrocycles and the most abundantpigments found in nature. They play a central role in electron transferdependent energy generating processes such as photosynthesis andrespiration. They further function as prosthetic groups for a variety ofenzymes, including catalases, peroxidases, cytochromes of the P450 classand sensor molecules. Heme is a hydrophobic molecule and associatesnon-specifically with lipids and proteins in aqueous solution where itpromotes peroxidations. Due to its hydrophobicity und toxicity, heme hasto be transported to its target proteins by different mechanisms, e.g.transport by transmembrane proteins, heme binding proteins and hemechaperones.We identified E. coli HemW as a potential heme-binding protein. Tocharacterize the heme-binding E. coli HemW was overproduced,anaerobically purified and a gel permeation chromatography wasperformed. Upon heme supplementation HemW dimerizes.First EPR spectra of E. coli HemW incubated with heme revealed anspectrum typical of an oxidized [4Fe-4S] 3+ cluster indicating electronBIOspektrum | Tagungsband 2012