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

alternative pathway for propionyl-CoA degradation must exist. To elucidatethe responsible pathway we performed several proteomic and microarraystudies. Interestingly, all experiments implied that propionyl-CoA isdegraded via beta-oxidation of fatty acids, although is has been assumed thatthe dehydrogenation of propionyl-CoA to acryloyl-CoA isthermodynamically unfavored. However, in agreement with the assumptionof beta-oxidation, a fox2 mutant, encoding for a 3-hydroxyacyl-CoAepimerase, required for fatty acid beta-oxidation, was unable to usepropionate as sole carbon and energy source. Surprisingly, growth testsshowed that the fox2 mutant is still able to use 3-hydroxypropionate as solecarbon source. Thus, it appears likely that 3-hydroxypropionate is anintermediate of a modified beta oxidation for propionyl-CoA degradationand the final product most likely consists of acetyl-CoA. To further confirmthis assumption, we are currently generating mutant strains of the postulatedbranch of the beta oxidation and apply NMR analyzes on C. albicans wildtype and mutant cells grown on 13 C-labeled propionate. Results will show,whether intermediates of a modified beta-oxidation of propionyl-CoAaccumulate in the respective mutants.PSP019Resting spores of Streptomyces coelicolor harbour anactive respiratory nitrate reductaseM. Fischer*, D. Falke*, G. SawersInstitute of Biology/Microbiology, Martin-Luther-University Halle-Wittenberg, Halle, GermanyStreptomyces coelicolor is an obligate aerobic soil bacterium that belongs tothe high-GC Gram-positive actinobacteria. A characteristic of this group is acomplex life cycle with stages that include vegetative hyphae, hydrophobicaerial hyphae and production of exospores. During spore formation specificstructural proteins, enzymes and storage compounds are synthesized andincorporated into the final spore compartment. These various cellularcomponents ensure that metabolism of these resting spores is maintained at alow-level to retain viability over long periods and at the same time allowsthem to survive a barrage of environmental insults. Long-term survivalrequires that essential metabolic pathways to cope with anaerobic conditionsare also present. The ability to respire with nitrate is one means by whichthis can be achieved. The genome of S. coelicolor has three narGHJIoperons, each encoding a respiratory nitrate reductase (Nar) [1], which ismembrane-associated with the active site facing the cytoplasm. Previousstudies have demonstrated that in spores and exponentially growingmycelium Nar-dependent nitrate reduction occurs [2].In this study we investigated which Nar is active in spores. Freshlyharvested spores of S. coelicolor wild type M145 could reduce nitrate at asignificant rate without addition of an exogenous electron donor. Moreover,this activity was also detectable in crude extracts of spores and could bevisualized by direct staining after native PAGE. Analysis of definedknockout mutants demonstrated that this activity was due to Nar1. Using adiscontinuous assay to measure nitrite production by spores we could showthat Nar1 was only capable of nitrate reduction under anaerobic conditions.Since Nar1 activity was measurable in crude extracts of spores that wereincubated both anaerobically and aerobically this finding suggests thatspores regulate either nitrate transport or Nar1 activity in response tooxygen. Notably, studies using protein synthesis inhibitors revealed thatNar1 is always present and active in resting spores.[1] van Keulen, G. et al (2005): Nitrate respiration in the actinomycete Streptomyces coelicolor.Biochem Soc Trans 33(Pt 1):210-2.[2] Fischer, M. et al (2010): The obligate aerobe Streptomyces coelicolor A3(2) synthesizes threeactive respiratory nitrate reductases. Microbiology 156(Pt 10):3166-79.PSP020Diversity in bacterial degradation of the steroidcompound cholateV. Suvekbala, J. Holert*, B. PhilippDepartment of Biology, Microbial Ecology, University of Konstanz,Konstanz, Germanybacteria was assessed by quantitative enrichments of steroid-degradingbacteria with littoral sediments of Lake Constance and the bile salt cholateas a model substance.Fifteen different strains of cholate-degrading bacteria were isolated fromhigh dilutions of littoral sediments. Two strains were characterized further.According to growth experiments and HPLC-analysis the first strain,Zoogloea sp. strain 1, degraded cholate via the 9,10-seco pathway asindicated by the formation of the characteristic degradation intermediatesDHADD (7,12-dihydroxy-1,4-androstadiene-3,17-dione) and THSATD(3,7,12-trihydroxy-9,10-secoandrosta-1,3,5(10)triene-9,17-dione). Duringcholate degradation by the second strain, Dietzia sp. strain 2, thecharacteristic intermediates of the 9,10-seco-pathway were not detected.Instead, two new compounds were detected by HPLC-analysis that differedfrom the UV-spectra of steroid compounds occurring in the 9,10-secopathway.Strain 2 could also not grow with the characteristic intermediatesof cholate degradation, which were isolated from cultures of the cholatedegradingbacterium Pseudomonas sp. strain Chol1 [2, 3]. In addition, thepresence of these compounds inhibited cholate degradation by strain 2.These results clearly showed that strain 2 harbours a different pathway forcholate degradation, which has not been described so far, indicating that thebiochemical diversity of aerobic steroid degradation in bacteria has beenunderestimated.[1] Philipp (2010): Appl Microbiol Biotechnol in press.[2] Birkenmaier et al (2007): J Bacteriol J Bacteriol 189:7165-7173.[3] Philipp et al (2006): Arch Microbiol 185:192-201.PSP021A novel high-affinity hydrogenase in Ralstonia eutrophaC. Schäfer*, A. Pohlmann, S. Frielingsdorf, O. Lenz, B. FriedrichInstitute for Biology, Microbiology, Berlin, GermanyWithin the global hydrogen cycle, soil deposition is the most importantnatural process responsible for removal of H 2 from the atmosphere.However, the mechanism by which H 2 is taken up remained elusive.Recently, a high-affinity hydrogenase has been indentified in spore-formingActinomycetes of the genus Streptomyces, which is able to oxidize H 2 atatmospheric levels. It has been suggested that this class of [NiFe]-hydrogenases is responsible for the H 2 uptake in soils [1].Interestingly, the genes coding for this high-affinity hydrogenase are alsopresent in the genome of the beta-proteobacterium Ralstonia eutropha [2].The two structural genes, encoded the hydrogenase small and large subunits,are part of a conserved operon structure, which also contains a complete setof hydrogenase maturation genes and a number of conserved unknowngenes. On the basis of its high similarity to the hydrogenases from sporeformingactinomycetes, the protein was designated actinomyceteshydrogenase (AH).Recently, we could show that Ralstonia eutropha cells containing solely theAH are capable in H 2 uptake as determined by gas chromatography. For adetailed investigation of the biochemical properties of the AH, a strain wasconstructed in which the weak native promoter of the AH operon wasexchanged by the strong promoter of the membrane-bound hydrogenasegenes from Ralstonia eutropha. AH-mediated H 2-oxidizing activity insoluble protein extracts was shown by activity staining in native gels usingNBT as an artificial electron acceptor. The AH was active also in theproduction of HD and D 2 from D 2O as shown by H/D-exchangeexperiments. We are currently constructing an AH derivative carrying anaffinity tag for facile purification and subsequent electrochemical andspectroscopic characterization. Furthermore, we are conducting experimentsin order to determine the regulatory background of AH gene expression andthe role of this interesting enzyme in R. eutropha. One attractive hypothesisis that the AH may contribute to the survival of the cells under starvationconditions by using the atmospheric trace concentrations of H 2.[1] Constant, P. et al (2010): Streptomycetes contributing to atmospheric molecular hydrogen soiluptake are widespread and encode a putative high-affinity [NiFe]-hydrogenase. Environ Microbiol.12(3), 821-9.[2] Schwartz, E. et al (2003): Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16megaplasmid encoding key enzymes of H(2)-based lithoautotrophy and anaerobiosis. J Mol Biol.332(2), 369-83.Steroids are ubiquitous natural compounds with diverse functions ineukaryotic organisms. They enter the environment mainly via excretion byand decay of animals and plants. In bacteria, steroids occur only as rareexceptions but the ability of transforming and degrading steroids iswidespread among bacteria. The only well-described pathway for aerobicdegradation of steroid compounds is the so-called 9,10-seco pathway [1]. Inthis study, the organismic and biochemical diversity of steroid-degradingspektrum | Tagungsband 2011