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

189or Mn 2+ were required for function. endA is co-transcribed with anextracellular phosphatase phoA and not upregulated uponphosphostarvation. Deletion of endA abolished extracellular degradation ofDNA by S. oneidensis MR-1 and the ability to use eDNA as sole source ofphosphorus. PhoA is not strictly required to exploit eDNA as nutrient. Theactivity of EndA prevents the formation of large cell aggregates duringplanktonic growth. However, in contrast to ExeM a deletion of endA hadonly minor effects on biofilm formation. The findings underline theimportance of extracellular nucleolytic activity for Shewanella andstrongly suggest specific functions for the different nucleases.PSP059Caffeate respiration in the acetogenic bacteriumAcetobacterium woodii: Characterization of a caffeateactivatingCoA transferaseV. Hess*, V. MüllerMolecular Microbiology & Bioenergetics, Institute for MolecularBiosciences, Goethe University, Frankfurt/Main, Germany, GermanyThe anaerobic acetogenic bacterium Acetobacterium woodii couples thereduction of caffeate with electrons derived from molecular hydrogen tothe synthesis of ATP by a chemiosmotic mechanism with Na + as couplingions. This process is called caffeate respiration (1). The Na + -translocatingenzyme in this respiratory pathway was identified as a ferredoxin:NAD + -oxidoreductase (Rnf) (2,3). Recently, the enzymes involved in caffeatereduction with electrons derived from the Rnf complex could be shown tobe encoded by the so called caffeate reduction operon carABCDE (4). Thefirst gene of the operon, carA, was annotated as a putative CoAtransferase. To further elucidate the function of CarA, the gene was clonedinto pET21a, heterologously overproduced in Escherichia coli and purifiedto apparent homogenity via IMAC. In a photometric assay, CarA of A.woodii could be affirmed as a hydrocaffeyl-CoA:caffeate CoA transferase.The biochemical properties of the enzyme are described and its role in thecaffeate reducing process is discussed.1) Müller, V., Imkamp, F., Biegel, E., Schmidt, S., Dilling, S. (2008) Discovery of aferredoxin:NAD + -oxidoreductase (Rnf) in Acetobacterium woodii: A novel potential coupling sitein acetogens. Ann. N.Y. Acad. Sci. 1125:137-1462) Imkamp, F., Biegel, E., Jayamani, E., Buckel, W. and Müller, V. (2007) Dissection of the caffeatrespiratory chain in the acetogen Acetobacterium woodii: Identification of an Rnf-type NADHdehydrogenase as a potential coupling site. J. Bacteriol. 189:8145-81533) Biegel, E. and Müller, V. (2010) Bacterial Na + -translocating ferredoxin:NAD + oxidoreductase.Proc. Natl. Acad. Sci. U .S. A. 107:18138-181424) Hess, V., Vitt, S. and Müller, V. (2011) A caffeyl-coenzyme A synthetase initiates caffeateactivation prior to caffeate reduction in the acetogenic bacterium Acetobacterium woodii. J.Bacteriol. 193:971-978PSP060A bacterial electron bifurcating uptake hydrogenaseK. Schuchmann*, V. MüllerMolecular Microbiology & Bioenergetics, Institute for MolecularBiosciences, Goethe University, Frankfurt/Main, Germany, GermanyA [FeFe]-hydrogenase containing four subunits (HydABCD) was purifiedfrom the cytoplasm of Acetobacterium woodii and the encoding geneswere identified. The complex is predicted to have one [H]-cluster, three[2Fe2S]- and six [4Fe4S]-clusters consistent with the experimentaldetermination of 32 mol of Fe and 30 mol of acid labile sulfur. Theenzyme catalyzed the exergonic reduction of NAD + with hydrogen asreductant only in the presence of flavin and ferredoxin. A k M, app for FMNof 6 M and for ferredoxin of 12 M was determined. The enzyme alsocatalyzed the endergonic reduction of ferredoxin with H 2 as reductant in areaction that was also strictly dependent on NAD + and FMN. Spectralanalyses revealed that ferredoxin and NAD + were reduced at the same timewith a stoichiometry of 1:1. Apparently, the multimeric hydrogenase of A.woodii used the novel mechanism of electron bifurcation in which theendergonic reduction of ferredoxin with electrons derived from molecularhydrogen is coupled with the exergonic electron transfer from molecularhydrogen to NAD + . The implications for the energy metabolism ofacetogenic and other bacteria are discussed.PSP061Initial insights into the organohalide respiratory process ofDehalococcoides sp. strain CBDB1A. Kublik*, C. Schipp, L. AdrianUFZ, Isotope Biogeochemistry, Leipzig, GermanyMicrobial reductive dechlorination plays a crucial role in the detoxificationof persistent halogenated compounds in contaminated environments.Several anaerobic bacteria like Dehalococcoides species are able to use awide range of these contaminants as terminal electron acceptors in ananaerobic respiration with hydrogen as the so far sole known electrondonor. This specialization is also reflected in the high number of putativereductive dehalogenase genes in the available genomes (e.g. 32 reductivedehalogenase homologous genes in Dehalococcoides sp. strain CBDB1 [1]).To understand this exceptional lifestyle, we aim to elucidate the respiratoryelectron-transfer chain between the hydrogenase(s) and the reductivedehalogenase(s) including the identification and characterization of theelectron-conducting components using Dehalococcoides sp. strain CBDB1as a model organism. We have assigned an enzymatic function for one ofthe reductive dehalogenase homologous genes using native gelelectrophoresis [2]. Also, we have now revised all cultivation, cellcounting, cell harvesting as well as protein quantification and proteinenrichment procedures to provide sufficient amounts of biomass foradvanced biochemical experiments. Correlation of protein expressionpatterns with different halogenated electron acceptors using enzymaticactivity tests and mass spectrometric analysis indicates the involvement ofa series of reductive dehalogenase proteins in various dehalogenationreactions. On the other hand, several potential membrane soluble electronconductingcandidates were identified and are further studied by in vitroenzyme activity assays. We also started with a general analysis of putativerespiratory chain components by liquid chromatography massspectrometry (LC-MS/MS) on the protein level.Acknowledgement: This work is supported by the DFG (Research Unit FOR1530).[1] Kube et al. (2005), Nature Biotechnol. 23: 1269-1273.[2] Adrian et al. (2007), Appl. Environ. Microbiol. 73: 7717-7724.PSP062Characterisation of a peptidyl-prolyl-cis-trans-isomerase ofCorynebacterium glutamicumN. Kallscheuer, J. van Ooyen*, T. Polen, M. BottForschungszentrum Jülich GmbH, IBG-1: Biotechnologie, Jülich, GermanySince the first report of the enzyme-driven cis-trans isomerization ofpetidyl-prolyl-bonds in polypetides, the peptidyl-prolyl-cis-transisomerases (PPIases) were found to be present in almost all sequencedgenomes to date. In eukaryotes PPiases play a major role in signaltransduction of the immune system, thus the mechanisms and inhibitors arewidely investigated. PPIases are classified in three distinct familiesreflecting their biochemical properties of binding to distinct classes ofinhibitor molecules. In prokaryotes, however, many PPIases wereinvestigated in vitro, but only in a few cases in vivo effects had beenpursued. We here present evidence for the in vivo function of theprokaryotic PPiase FkpA of the soil bacterium and model microorganismCorynebacterium glutamicum. FkpA belongs to the family of FK-506binding proteins (FKBPs) which are inhibited by FK-506 (Tacrolimus).At temperatures below 25°C and above 35°C FkpA has a positive effect oncitrate synthase (CS) activityin vitroand delayed aggregation of CS at 37°Cand above. In vivo, deletion of fkpA leads to decreased cell growth and thespecific CS activities of C. glutamicumfkpA were found to be reduced byabout 40%, when cells were cultivated in synthetic media containing eitherglucose or acetate as carbon source. DNA microarray analyses comparingthe transcriptomes of C. glutamicumfkpA and the wild type revealed,amongst others, that the RNA level of lactate dehydrogenase (ldh) was 10-fold increased in the deletion mutant, whereas the specific Ldh-activitywas solely slightly increased, which could be interpreted as a direct effectof missing PPiase activity. Taken together we provide evidence thatPPiases play a major role in protein stability and folding and therebymodulate enzyme activity in prokaryotes. Additionally, PPiases maybroaden the optimal temperature range of their substrate enzymes.PSP063Differentiation of respiratory molybdopterin-containingoxidoreductases: insight multiple functions, structures andgenetic compositionO. KlimmekInstitute of Microbiology + Genetics, Department of Biology, Darmstadt,GermanyBacteria commonly perform anaerobic respiration driven by electrontransport chains. Such respiratory chains consist of membrane-bound andsoluble electron transport proteins that are involved in proton motive forcegeneration. A high number of these proteins are molybdopterin-containingoxidoreductases that belong to the dimethyl sulfoxide (DMSO) reductasefamily. From an evolutionary point of view, it is assumed that thesemolybdoproteins are ancient enzymes involved, for example, in conversionof sulfur containing compounds.Due to the homology within this group of molybdoenzymes it ischallenging to predict the substrate range of such enzymes from genomedata only. In particular, high similarities exist within the hydrophilicsubunits of the DMSO reductase family comprising catalytic subunitscontaining the MGD ligated molybdenum ion as well as electron transfersubunits containing Fe/S clusters. In contrast the quinone- / quinol-reactivemembrane subunits show differences [1,2].The aim of this study was to functionally discriminate molybdoenzymescontaining membrane subunits of the PsrC/NrfD family. The genome ofthe model organism Wolinella succinogenes encodes 11 differentrespiratory molybdoenzymes [3], several of which have unknownfunctions. Five of them are predicted to contain subunits of the PsrC/NrfDBIOspektrum | Tagungsband 2012