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

AMV008Structure and function of the SAM-dependenturoporphyrinogen III methyltransferase NirE involved inheme d 1 biosynthesis in Pseudomonas aeruginosaS. Storbeck, G. Layer*Institute for Microbiology, University of Technology, Braunschweig,GermanyAnaerobic growth and survival of Pseudomonas aeruginosa is essential forbiofilm formation and infection. Replacement of the electron acceptoroxygen by nitrate during denitrification is a powerful strategy for anaerobicenergy generation and ecologically indispensable for the global nitrogencycle. In the second step of the denitrification process the dissimilatorynitrite reductase (cytochrome cd 1) utilizes the prosthetic groups heme c andheme d 1 for the reduction of nitrite to nitric oxide. Heme d 1 is not a realheme, rather an isobacteriochlorin related to siroheme, vitamin B 12 andcoenzyme F 430. The multistep biosynthesis of this unique cofactor is onlypoorly understood. The SAM-dependent uroporphyrinogen IIImethyltransferase NirE catalyzes the key branchpoint step of heme d 1biosynthesis, namely the methylation of uroporphyrinogen III to precorrin-2.We produced and purified recombinant NirE from P. aeruginosa. PurifiedNirE was biochemically characterized showing for the first time that thisprotein carries SAM-dependent uroporphyrinogen III methyltransferaseactivity. The crystal structure of NirE was solved in complex with itssubstrate uroporphyrinogen III and the reaction product S-adenosylhomocysteine. The role of conserved amino acid residuespotentially involved in the catalytic mechanism was investigated by sitedirected mutagenesis. Based on the structure of the enzyme-substratecomplex and the mutagenesis studies we propose a novel reactionmechanism for the NirE catalyzed reaction involving a highly conservedarginine residue as the catalytically essential base.AMP001The ptx-ptd locus from Desulfotignum phosphitoxidanshas a dual function in phosphite metabolism of this strainD. Simeonova*, B. SchinkDepartment of Biology, University of Konstanz, Konstanz, GermanyPhosphorus in living systems typically exists in the [+5] oxidation state asphosphate, phosphate esters, or phosphate anhydrides. Several aerobicbacteria are able to oxidize phosphite [+3] to phosphate [+5] incorporatingthe latter into their biomass. The first proof of phosphite oxidation as a typeof energy metabolism was found with the isolation of an anaerobicphosphite-oxidizing sulfate-reducing bacterium, Desulfotignumphosphitoxidans [1].A genomic library of D. phosphitoxidans was screened for clones harboringa gene coding for a protein in the proteome of the strain that is induced byphosphite [2]. Sequence analysis of two positive clones revealed an operonof seven genes ptxED-ptdFCGHI predicted to be involved in phosphiteoxidation. Four of these genes (ptxD-ptdFCG) were cloned andheterologously expressed in Desulfotignum balticum, a related strain thatcannot use phosphite as either an electron donor, or as a phosphorus source.The four-gene cluster was sufficient to confer phosphite uptake andoxidation ability to the host strain [3]. Therefore the ptx-ptd cluster from D.phosphitoxidans plays a double role in phosphite metabolism in this strain, -once in the energy metabolism where phosphite serves as electron donor andsecond in the supplementation of the strain with phosphorus source forassimilation when needed.[1] Schink, B. et al (2002): Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer thatoxidizes phosphite to phosphate. Arch Microbiol 177:381-391.[2] Simeonova D.D. et al (2009): Unknown-genome-proteomics. A new NAD(P)-dependentepimerase/ dehydratase revealed by N-terminal sequencing, inverted PCR and high resolution massspectrometry. Mol Cell Proteomics 8 (1): 122-131.[3] Simeonova D.D. et al (2010): Identification and heterologous expression of genes involved inanaerobic dissimilatory phosphite oxidation by Desulfotignum phosphitoxidans. J Bacteriol, 192 (19):5237-5244.AMP002Development of a Genetic System for GeobactermetallireducensJ. Oberender*, M. BollInstitute for Biochemistry, University of Leipzig, Leipzig, GermanyMembers of the obligately anaerobic, metal oxide respiring genus Geobacterplay an important role in the bioremediation of organic compounds [1].Growth substrates of Geobacter species include various aromaticcompounds like benzoate, phenol, p-cresol and toluene. Recent studiesrevealed that obligately anaerobic bacteria such as G. metallireducens andfacultative anaerobes use different key enzymes for the completedegradation of aromatic growth substrates [2]. To open the door for studyingthe role of unknown gene products in aromatic degradation pathways, agenetic system was established for G. metallireducens. The antibioticsensitivity of this organism was characterized and conditions for efficientcultivation on solid medium were established. A procedure for introducingforeign DNA by electrotransformation was developed. The broad-host rangevector pCD342 [3] was used for homologous expression of bamY, the onlygene in the genome that was predicted to code for a benzoate-CoA ligase.This enzyme activates benzoate to benzoyl-CoA, the central intermediate ofmost anaerobic aromatic degradation pathways [4]. Mutants of G.metallireducens with a disrupted bamY gene were surprisingly still able touse benzoate as the sole carbon source. The presence of an unorthodoxbenzoate-CoA ligase or benzoyl-CoA:acceptor carboxylic acid CoAtransferase is being studied.[1] Lovley et al (1993): Arch Microbiol. 159:336-344.[2] Kung et al (2009): PNAS. 106(42):17687-176892.[3] Dehio et al (1998): Gene. 215:223-229.[4] Wischgoll et al (2005): Mol Microbiol. 58(5):1238-1252.AMP003Microbial reduction of Fe oxides at low ionic strengthJ. Braunschweig*, J. Bosch, R.U. MeckenstockInstitute of Groundwater Ecology, Helmholtz Center Munich, Neuherberg,GermanyMicrobial iron reduction is a major biogeochemical process in groundwaterecosystems and often associated with the degradation of organiccontaminants. Iron reduction is limited by the high crystallinity and lowsolubility of iron oxides which can be overcome by the use of electronshuttles like humic substances [2]. Furthermore, a recent study showed thatcatalytic amounts of ferrihydrite colloids added to bulk ferrihydrite lead tothe complete reduction of iron oxides by Geobacter sulfurreducens [1].The objective of this work was to inquire if adsorbed organic moleculespassivate the colloid surfaces or stimulate the catalytic effect of colloidaliron oxides. Microbial anaerobic reduction experiments with G.sulfurreducens were conducted with 260 nm ferrihydrite colloids in a 100-fold diluted freshwater medium. Acetate was used as model organiccompound. Within the first 30 hours, the ferrihydrite was totally reduced.This high reactivity is attributed to the high spatial availability of thenanosized ferrihydrite colloids and therefore a higher bioavailability thanbulk ferrihydrite. During sorption experiments with ferrihydrite colloids andfulvic acids from Gorleben the sorption capacity was determined.In conclusion, nanosized iron oxides are supposed to play a significant rolein electron transfer processes in anoxic ecosystems.[1] Bosch, J. et al (2010): Nanosized iron oxide colloids strongly enhance microbial iron reduction.Appl. Environ. Microbiol. 76, 184-189.[2] Lovley, D. R. et al (1996): Humic substances as electron acceptors for microbial respiration.Nature 382, 445-448.AMP004Function and Regulation of Carbon MonoxideDehydrogenase/Acetyl-CoA Synthase in MethanosarcinaacetivoransN. Matschiavelli*, E. Oelgeschläger, M. RotherInstitute for Molecular Bio Science, Goethe-University, Frankfurt am Main,GermanyMethanosarcina species are among the most metabolically versatilemethanogens, as they can use methylated compounds, H 2+CO 2 or acetate forgrowth as well. The model organism Methanosarcina acetivorans, a marinemesophile, is unable to utilize H 2+CO 2, but can use carbon monoxide (CO)spektrum | Tagungsband 2011