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

186potential active site region. We demonstrated the ability of Mcup_0683 to bindsulfur via MALDI-TOF mass spectrometry. These findings provide strongmotivation to investigate the potential of proteins Mcup_0681-0683 to acttogether in a sulfur relay system involved in dissimilatory sulfur oxidation inMetallosphaera cuprina and possibly also in other archaea and bacteria.1. Liu, L.J., et al. 2011. Int. J. Syst. Evol. Microbiol.,61: 2395-2400.2. Liu, L.J., et al. 2011. J. Bacteriol.,193: 3387-3388.3. Ikeuchi, Y., et al. 2006. Mol. Cell,21: 97-108.4. Dahl, C., et al. 2008. J. Mol. Biol,384: 1287-1300.5. Auernik, K., et al. 2008. Appl. Environ. Microbiol.,74: 7723-7732.6. Quatrini, R., et al. 2009. BMC Genomics,10: 394.PSP045Elucidation of the Periplasmic Cytochrome Network inShewanella oneidensis MR-1G. Sturm*, J. GescherKarlsruher Institut für Technologie, Angewandte Biologie, Karlsruhe, GermanyShewanella oneidensis MR-1 is a Gram-negative soil bacterium whichshows an astonishing versatility in terms of electron acceptors it can use.The predominant proteins driving respiratory electron transfer from thecytoplasm to periplasmic space and from there to the outer membrane arec-type cytochromes. Interestingly, S. oneidensis cells express a largenumber of periplasmic c-type cytochromes that are not primarily involvedin iron reduction (i.e. SoxA-like, NrfA and CcpA) even when they growunder iron reducing conditions. Furthermore, our experiments revealedthat iron grown cells are able to conduct electron transfer to a multitude ofelectron acceptors although they had not been in contact to one of theseacceptors in the growth medium. It seems fairly possible that theseperiplasmic c-type cytochromes build up a network which allows electronexchange between respiratory pathways. This feature would certainlyenable the cell to quickly respond to changes in the availability of electronacceptors that occur in its environment. Examples for connected respiratorypathways will be presented. Still, although it is generally believed that c-typecytochromes conduct rather unspecific electron transfer it was possible to showthat is not necessarily the case. The electron transport pathway to the peroxidaseCcpA is an example for specificity within c-type cytochrome dependentelectron transfer. The two cytochromes involved, CcpA and ScyA, aredisconnected from other pathways. CcpA functions as a peroxidase protectingthe cell against oxidative stress caused by hydrogen peroxide possibly producedduring dissimilatory iron reduction via the Fenton reaction. CcpA gains itselectrons exclusively from ScyA, a small monoheme cytochrome. In this studythe range and dynamic of the periplasmic c-type cytochrome network will bepresented in further detail.PSP046Complete -oxidation of the acyl side chain of cholate byPseudomonas sp. strain Chol1 in vitroJ. Holert* 1 , O. Yücel 2 , Ž. Kuli 3 , H. Möller 3 , B. Philipp 11 WWU Münster, IMMB, Münster, Germany2 University of Konstanz, Biology, Konstanz, Germany3 University of Konstanz, Chemistry, Konstanz, GermanySteroids are ubiquitous natural compounds with diverse functions ineukaryotes. In bacteria, steroids occur only as rare exceptions but the ability oftransforming and degrading steroids is widespread among bacteria.We investigate bacterial steroid degradation using the bile salt cholate as amodel compound and Pseudomonas sp. strain Chol1 as a model organism.Cholate degradation is initiated by oxidative reactions at the A-ringfollowed by cleavage of the side chain attached to C17. Mutants of strainChol1 with defects in the genes skt and acad are defect in the degradationof the acyl side chain. In culture supernatants of these mutants, (22E)-7,12-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO) and7,12-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC),respectively, accumulate as dead end products. The structure of thesecompounds indicates that degradation of the acyl side chain proceeds via-oxidation but explicit in vitro data was missing so far. We investigatedthe degradation of the acyl side in vitro using cell extracts of strain Chol1in the presence of co-factors (CoA, ATP, NAD + andphenazinemetholsulfate). When cholate or 1,4 -3-ketocholate were used assubstrates, 1,4 -3-ketocholyl-CoA was the end product indicating thatfurther oxidation of the acyl side chain was not possible in vitro under theapplied conditions. When either DHOCTO or DHOPDC were used thecomplete side chain was cleaved off in vitro leading to 7,12-dihydroxyandrosta-1,4-diene-3,17-dione(12-DHADD) as end product. With bothsubstrates the CoA-ester of DHOPDC accumulated transiently in the assay.With cell extracts of the skt mutant DHOCTO was converted toDHOCTO-CoA which was not further degraded to 12-DHADD. Withcell extracts of the acad mutant DHOCTO was converted to DHOPDC-CoA, which was also not further degraded. Thus, the phenotypes of bothmutants were confirmed by these in vitro assays.To our knowledge this is the first detailed in vitro demonstration of thecomplete degradation of a steroid side chain by -oxidation in bacteria.Furthermore, our results indicate that the dehydrogenation reactions of 1,4 -3-ketocholyl-CoA and of DHOPDC-CoA are the rate limiting steps inthis -oxidation pathway.PSP047Genomic plasticity responsible for dissimilatory iron reductionin Shewanella oneidensis MR-1S. Stephan*, M. Schicklberger, J. GescherKarlsruher Institut für Technologie, Angewandte Biologie, Karlsruhe, GermanyThe ability of the facultative anaerobic bacterium Shewanella oneidensisMR-1 to respire poorly soluble electron acceptors under anoxic conditionsrelies on a complex electron transfer network. Four distinct pathwayspredicted to facilitate respiratory electron flow to extracellular electronacceptors are encoded in the genome of S. oneidensis MR-1. Thesepathways share MtrA (metalreducing protein A) and MtrB paralogues,which are periplasmic c-type chytochromes and integral outer membranebeta-barrel proteins, respectively (1). Interestingly gene clusters encodingMtrA and MtrB homologs are phylogenetically distributed among allclasses of proteobacteria and the corresponding proteins were shown to benot only involved in ferric iron reduction but also ferrous iron oxidation (2).A mtrB null mutant strain in Shewanella lacks the ability to respire onFe(III)-oxides (3). Interstingly, after prolonged incubation supressormutations occur that rescue the mutant phenotype. In this work we isolatedand characterized such a mtrB suppressor mutant. Molecular and geneticanalysis revealed that the suppression relies on a functional replacement ofMtrB and MtrA by homologous proteins encoded by SO4359 and SO4360respectively. This replacement underlies a transcriptional upregulation ofthe SO4362-SO4357 gene cluster which was found to be due to aninsertion sequence (ISSod1) belonging to the IS-1 superfamily generatinga constitutively active hybrid promoter. Here we could show for the first time afunctional replacement of the MtrAB subcomplex by a complex consistent ofhomologous proteins and the involvment of SO4360 as periplasmic electroncarrier in dissimilatory iron reduction in Shewanella oneidensis MR-1.(1) Gralnick JA, Vali H, Lies DP, Newman DK (2006): Extracellular respiration of dimethylsulfoxide by Shewanella oneidensis strain MR-1.(2) Jiao Y, Newman DK (2007): ThepioOperon Is Essential for Phototrophic Fe(II) Oxidation inRhodopseudomonas palustris TIE-1.(3) Beliaev AS, Saffarini DA (1998): Shewanella putrefaciens mtrB Encodes an Outer MembraneProtein Required for Fe(III) and Mn(IV) Reduction.PSP048The phosphotransferase system CAC0231-CAC0234 controlsfructose utilization of Clostridium acetobutylicumC. Voigt*, H. Janssen, R.-J. FischerInstitute of Biological Sciences/University of Rostock, Division ofMicrobiology, Rostock, GermanyClostridium acetobutylicum is well characterized by its biphasicfermentation metabolism. At higher pH values exponentially growing cellsusually produce acetate and butyrate as main fermentation productswhereas when the pH has dropped below 5.0 the metabolism switches to‘solventogenesis’ with the dominating fermentation products butanol andacetone. As a carbon and energy source a variety of carbohydrates likeglucose, fructose or xylose can be utilized by C. acetobutylicum.Generally, carbohydrates were taken up via three types of transporters:symporter, ATP-binding cassette (ABC) transporter andphosphotransferase systems (PTS). For the uptake of hexoses, hexitols anddisaccharides thirteen PTS have been identified in C. acetobutylicum.Among them, three PTS are supposed to be responsible for the uptake offructose. The apparent primary fructose transport system is encoded by apolycistronic operon (cac0231-cac0234) including a putative DeoR-typetranscriptional regulator (CAC0231), a 1-phosphofructokinase(CAC0232), a PTS IIA (CAC0233) and a PTS IIBC (CAC0234). Toanalyze the role of the PTS during growth on fructose as sole carbonsource, each single gene of the operon (cac0231-cac0234) was specificallyinterrupted using the ClosTron® system. All mutant strains showedimpaired growth due to reduced fructose consumption. Interestingly, aconcomitant loss of solvent production was monitored indicating athreshold of sugar concentration for initiation of the metabolic switch.Moreover, the transcriptional regulator CAC0231 was overexpressed in E.coli and purified for electrophoretic mobility shift assays (EMSA). Here, aputative binding motif was identified and proved by a specific binding ofCAC0231 to the promoter region of cac0231-cac0234.BIOspektrum | Tagungsband 2012