VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

The gene product of PA1242 (sprP) contains a predicted signal sequenceand a peptidase S8 domain. However, it contains a non-canonical catalytictriad composed of histidine, asparagine and serine. Sequence analysisrevealed the presence of an additional element in the domain organization ofthe protease. SprP carries beside its signal peptide and the S8 domain adomain of unknown function (DUF) between both elements. Afteridentification SprP was cloned, expressed in E. coli and the protease activitywas measured with protease substrates like casein and Suc-AAPF-pNA.Proteases have also an impact on different physiological processes likeprotein processing and activation, secretion of other proteins andpathogenicity of the host bacterium. A P. aeruginosa sprP-negative mutantwas constructed and different phenotypes were tested to elucidate thephysiological role of SprP. We were able to illustrate an eminent role ofSprP by characterization of different phenotypes. Deletion of sprP causesthe loss of motility, an increased biofilm formation and the accumulation ofcell aggregates during growth.PSP015The lipase specific chaperone LipH is required for properinner membrane translocation of Lipase A inPseudomonas aeruginosaR. ABOUBI* 1 , S. Wilhelm 1 , K.-E. Jäger 1 , F. Rosenau 21 Institute for Molecular Enzyme Technology, Heinrich-Heine-University,Jülich, Germany2 Institute for Pharmaceutical Biotechnology, Ulm, GermanyFolding of lipase A from P. aeruginosa essentially requires in vivo and invitro the action of the steric chaperone LipH. Such lipase specific foldases(Lif) consist of an amino terminal membrane anchor followed by a variable40 aa domain and the large carboxy terminal folding domain. In vitrorefolding experiments revealed that only the folding domain of LipH isneeded to fold lipases into their enzymatically active conformation. The 3Dstructure of such a folding domain, of the closely related lif protein fromBurkholderia glumae, was solved in complex with its cognate lipase. In thisstructure the variable region could not be modeled and was thereforesuggested to be very flexible or unstructured. A physiological function ofthis domain is unknown at present.We constructed a LipH variant, in which the variable domain was deleted.As consequence the N-terminal membrane anchor was directly attached tothe folding domain of LipH. Upon expression of this modified LipHtogether with its cognate lipase LipA in the homologous host P. aeruginosaa complete loss of secretion was detected. Not only lipase LipA was nolonger secreted but also other extracellular Sec substrates proteins such asElastaseB and ExotoxinA failed to reach the culture supernatant, whereasTAT substrates like phospholipase where perfectly secreted.Expression of the lipase together with the truncated foldase in P. aeruginosaleads to a blockage of the Sec apparatus and thus suggests a probablefunction of the variable domain for interaction of the protein with the Secapparatusthereby probably being involved in the release of lipase from theSec machinery.PSP016Biosynthesis and occurence of open chain tetrapyrroles incryptophytesK. Overkamp* 1 , J. Wiethaus 1 , K. Hoef-Emden 2 , N. Frankenberg-Dinkel 11 Physiology of Microorganisms, Ruhr-University, Bochum, Germany2 Institute of Botany, University of Cologne, Cologne, GermanyPhycobiliproteins are light-harvesting proteins, which occur incyanobacteria, red algae and cryptophytes in addition to chlorophyllcontaining antenna complexes. They allow the organisms to efficientlyabsorb light in regions of the visible spectrum that are poorly covered bychlorophylls. Cryptophytes are unicellular, eukaryotic algae and widespreadin marine and limnic waters. Their phycobiliproteins consist of an (αα‘ββ)heterotetrameric apo-protein covalently associated with characteristic openchain tetrapyrroles, which act as light absorbing chromophores.Cryptophytes employ the six different chromophores phycocyanobilin(PCB), phycoerythrobilin (PEB), 15,16 dihydrobiliverdin (15,16-DHBV),mesobiliverdin (MBV), bilin 584 and bilin 618 for light-harvesting.The chromophore composition of the novel phycobiliproteins PC577 andPC630 from the cryptophytes Hemiselmis pacifica and Chroomonas sp. isstill unknown. Purification of those phycobiliproteins and subsequentanalysis of isolated chromopeptides using High Performance LiquidChromatography (HPLC) and UV-Vis spectroscopy identified severalcandidate chromophores. While the PC630 α and α‘ subunits seem to beassociated with biliverdin IXα, the chromophore of the PC577 α and α‘subunit is still unknown. In contrast, PEB is most likely attached to the βsubunits of both proteins. Continuative HPLC and NMR experiments will bedone to elucidate the correct chromophore composition, which will givefurther insights into the evolutionary history of cryptophytes.Not only the chromophore composition of several phycobiliproteins incryptophytes is unknown but also the biosynthetic pathway of the openchain tetrapyrroles. Therefore the cryptophyte Guillardia theta in which thephycobiliprotein PE545 is associated with the chromophores 15,16-DHBVand PEB will serve as a model organism for the elucidation of thebiosynthetic pathway. Extensive bioinformatic analyses and amino acidsequence alignments identified a putative heme oxygenase and a PebB-likebilin reductase in G. theta. Currently, the enzymatic activities of theseputative bilin biosynthesis enzymes is investigated and compared to knownactivities of cyanobacteria and higher plants.PSP017Bacterial cytochrome c peroxidase BCCP of Shewanellaoneidensis Structure and physiological role underdissimilatory iron reducing conditionsB. Schütz* 1 , J. Seidel 2 , O. Einsle 2 , J. Gescher 11 Institute for Biology II/Microbiology, Albert-Ludwigs-University, Freiburg,Germany2 Institute for Organic Chemistry and Biochemistry, Albert-Ludwigs-University, Freiburg, GermanyBacterial diheme c-type cytochrome peroxidases (CcpAs) catalyze theperiplasmic reduction of hydrogen peroxide to water. The γ-proteobacteriumS. oneidensis produces the peroxidase BCCP under dissimilatory ironreducing conditions. We wanted to understand the function of this protein inthe organism as well as its putative connection to the electron transport chainto ferric iron. BCCP was isolated after heterologous expression and testedfor its peroxidase activity as well as for its structural conformation asanalyzed by X-ray crystallography. BCCP exhibited in vitro peroxidaseactivity and had a structure typical for diheme peroxidases. It was producedin almost equal amounts under anaerobic as well as microaerophilicconditions. With 50 mM ferric citrate and 50 μM oxygen in the growthmedium, BCCP expression results in a strong selective advantage for thecell as was detected in competitive growth experiments between wild typeand ΔccpA mutant cells that lack the entire ccpA gene due to a markerlessdeletion. This was expected since we observed a large fraction of theavailable oxygen being converted into hydrogen peroxide. Hydrogenperoxide production occurred during the entire time course of the growthexperiment and was apparently not coupled to a specific growth phase. Wewere unable to reduce BCCP directly with either CymA, MtrA or FccA butisolated the small monoheme ScyA as an electron transport mediatorbetween CymA and BCCP. As we also were unable to reduce ScyA withother periplasmic cytochromes CymA, ScyA and BCCP seem to build aspecific electron transport chain to hydrogen peroxide. Consequently, the sofar believed lack of specificity in interprotein electron transport between c-type cytochromes has to be questioned.PSP018Detoxification of propionyl-CoA in Candida albicans:Implications for a modified beta-oxidation pathwayC. Otzen*, M. BrockDepartment of Microbial Pathogenicity Mechanisms, Hans Knöll Institute(HKI), Jena, GermanyPropionyl-CoA is a common metabolite deriving from amino aciddegradation or breakdown of odd-chain fatty acids. All cells need to avoidan accumulation of propionyl-CoA, since this CoA-ester can interfere withvarious enzymatic reactions of primary carbon metabolism. Mammals andseveral bacteria use the so-called methylmalonyl-CoA pathway fordetoxification and metabolism of propionyl-CoA, leading to the citric acidcycle intermediate succinyl-CoA. Contrarily, some bacteria and most fungiutilize the methylcitrate cycle for propionyl-CoA degradation. In the latterpathway propionyl-CoA is alpha-oxidized and yields pyruvate. Interestingly,Candida albicans neither contains genes encoding enzymes of themethylmalonyl-CoA nor of the methylcitrate cycle, but is able to grow onpropionate, odd-chain fatty acids and proteins as carbon sources. Thus, anspektrum | Tagungsband 2011