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

OTP045Penicillin binding protein 2x of Streptococcuspneumoniae: A GFP-PBP2x fusion is functional andlocalizes at the division septumK. Peters*, C. Stahlmann, R. Hakenbeck, D. DenapaiteDepartment of Microbiology, University of Kaiserslautern, Kaiserslautern,GermanyPenicillin-binding protein 2x (PBP2x) is one of the six PBPs in S.pneumoniae involved in late steps of peptidoglycan biosynthesis. PBP2xcatalyse a penicillin-sensitive transpeptidation reaction. Mutations in PBP2xthat interfere with beta-lactam binding are crucial for the development ofhigh level penicillin-resistance which involves other PBPs as well. ThePBP2x gene is located in a cluster devoted to cell division, and localizationof PBP2x at the septum as revealed by immunofluorescence techniquesconfirmed its role in the division process [1]. However, immunostaining hasthe disadvantage that cells need to be fixed and have to undergo a damagingcell wall permeabilization treatment. Green fluorescence protein (GFP)fusions can overcome these problems and allow the visualization of fusionproteins in living cells.To investigate the role of PBP2x during growth and division of S.pneumoniae cells, an N-terminal GFP-PBP2x fusion was constructed usingplasmid pJWV25 that contains Zn 2+ -inducible promoter driving gfp-fusiongene expression [2]. This plasmid also carries the flanking regions of thenonessential S. pneumoniae bgaA gene, facilitating a double cross-overevent at this locus. GFP-PBP2x signal was observed at the septum in S.pneumoniae cells. Furthermore, the native copy of pbp2x gene could bedeleted in these cells without affecting cell growth, showing that GFP-PBP2x is functional. This system was applied to study cellular localizationof PBP2x protein in strains which contain a reduced amount of PBP2x.[1] Morlot, C. et al (2003): Growth and division of Streptococcus pneumoniae: localization of the highmolecular weight penicillin-binding proteins during the cell cycle. Mol Microbiol. 50(3): 845-55.[2] Eberhardt, A. et al (2009): Cellular localization of choline-utilization proteins in Streptococcuspneumoniae using novel fluorescent reporter systems. Mol Microbiol. 74(2): 395-408.OTP046Purification of the MCAP 3-halogenase from pyrrolnitrinbiosynthesis in P. fluorescens BL915A. Adam*, K.-H. van PéeDepartment of Biochemistry, University of Technology, Dresden, GermanyPyrrolnitrin is an antifungal compound [1] first isolated from Pseudomonaspyrrocinia [2]. The gene cluster responsible for pyrrolnitrin biosynthesiswas identified in Pseudomonas fluorescens (BL915) [3, 4] and otherpyrrolnitrin producing bacteria. Four conserved enzymes are involved inpyrrolnitrin biosynthesis, named PrnA, PrnB, PrnC, and PrnD, according totheir order in catalysis. The tryptophan 7-halogenase PrnA catalyzes theregioselective chlorination of the amino acid tryptophan in 7 position of theindole ring [6]. The second enzyme, PrnB, converts 7-Cl-tryptophan intomonodechloroamino-pyrrolnitrin [4]. This intermediate is chlorinated by thethird enzyme, PrnC, a second flavin-dependent halogenase. The fourthenzyme, PrnD, oxidizes the amino group to a nitro group, yieldingpyrrolnitrin [7].FADH 2-depending halogenases contain two conserved regions - theGxGxxG and the WxWxIP motif, leading to the assumption that the MCAP3-halogenase PrnC operates by the same mechanism as the well-analyzedtryptophan 7-halogenase PrnA. So far, the MCAP 3-halogenase PrnC couldnot be purified in active form, precluding further analysis. We now report anovel purification strategy leading to purified and active PrnC. Using theGST-fusion protein strategy it is possible to obtain pure PrnC produced by arecombinant Escherichia coli strain. Both, fusion protein and cleavedMCAP 3-halogenase show halogenating activity.[1] van Pée, K. H. and J. M. Ligon (2000): Nat. Prod. Rep., 17, 157-164.[2] Arima, K. et al (1964): Agric. Biol. Chem., 28, 575-576.[3] Hammer, P. E. et al (1997): Appl. Environ. Microbiol., 63, 2147-2154[4] Kirner, S. et al (1998): J. Bacteriol., 180, 1939-1943.[5] Hohaus, K. et al (1997): Angew Chem. Int. Ed. Engl., 36, 2012-2013.[6] Lee, J. K. et al (2006): J. Bacteriol., 188, 6179-6183.OTP047Flavoenzymes of Escherichia coli as targets for theriboflavin analog roseoflavin from StreptomycesdavawensisS. Langer*, S. Naganishi, M. MackMannheim University of Applied Sciences, Mannheim, GermanyThe gram-positive soil bacterium Streptomyces davawensis is the onlyknown organism to produce the antibiotic roseoflavin (8-dimethylamino-8-demethyl-D-riboflavin) a riboflavin (vitamin B 2) analog (4). Roseoflavinexhibits antibiotic activity against gram-positive and also gram-negativebacteria if a flavin uptake system is present (2). In the cytoplasm roseoflavinis converted to roseoflavin-5’-monophosphate (RoFMN) and roseoflavinadenine dinucleotide (RoFAD) by the combined activity of flavokinase (EC2.7.1.26) and FAD synthetase (EC 2.7.7.2) (1). A recombinant Escherichiacoli strain overproducing the flavin transporter PnuX (fromCorynebacterium glutamacium) is roseoflavin sensitive. Bacillus subtilisnaturally contains a flavin transporter and thus is roseoflavin sensitive aswell. Both bacteria were cultivated in the presence of riboflavin andsublethal amounts of roseoflavin. The total protein was isolated andanalyzed with respect to its flavin content. The total protein obtained fromriboflavin grown cells contained FMN and FAD, the total protein obtainedfrom roseoflavin grown cells in addition contained RoFMN. RoFAD wasnot detected.Subsequently, 40 different recombinant E. coli strains each overproducinganother his 6-tagged E. coli flavoenzyme were obtained through the ASKAlibrary (3). The flavoenzymes were synthesized in a PnuX overproducing E.coli strain in the presence of roseoflavin, purified by affinitychromatography and it was found that they contained RoFMN.It was reported that some enzymes are inactive in their roseoflavin cofactorform e.g. D-amino acid oxidase from Sus scrofa (RoFAD) (EC 1.4.3.3).Exemplarily, AzoR an azobenzene reductase (EC 1.7.1.6) from E. colinaturally containing FMN was purified in its FMN and RoFMN form.Present results indicate a decrease in activity up to 90%. All in all, we couldshow that roseoflavin was converted to RoFMN in vivo and that this flavinanalog was accepted as a cofactor by flavoenzymes of E. Coli which seemsto result in a loss off activity.[1] Grill, S., S. Busenbender, M. Pfeiffer, U. Kohler, and M. Mack. 2008. J Bacteriol 190:1546-53[2] Grill, S., H. Yamaguchi, H. Wagner, L. Zwahlen, U. Kusch, and M. Mack. 2007. Arch Microbiol188:377-87[3] Kitagawa M., A. Takeshi, M. Arifuzzaman, T. Ioka-Nakamichi, E. Inamoto, H. Toyonaga, H.Mori. 2005. DNA Research 12:291-299[4] Otani, S., M. Takatsu, M. Nakano, S. Kasai, and R. Miura. 1974. J Antibiot (Tokyo) 27:86-7.OTP048Managing Zoonotic Diseases - Research NetworkingG. Benninger* 1 , I. Semmler 2 , S.C. Semler 3 , A. Wiethölter 4 , M.H. Groschup 5 ,S. Ludwig 61 National Research Platform for Zoonoses, c/o Westphalian Wilhelms-University, Münster, Germany2 National Research Platform for Zoonoses, c/o TMF e.V, Berlin, Germany3 TMF e.V, Berlin, Germany4 National Research Platform for Zoonoses, c/o Friedrich Loeffler Institute,Greifswald - Insel Riems, Germany5 I nstitute for Novel and Emerging Infectious Diseases, Friedrich LoefflerInstitute, Greifswald - Insel Riems, Germany6 Institute of Molecular Virology, Wilhelms-University, Münster, GermanyZoonoses are infectious diseases which are transmitted from animals tohumans and vice-versa. They are caused by different types of agents -bacteria, parasites, fungi, prions or viruses. Over 200 zoonoses have beendescribed and the number is still increasing as new biomedical knowledge isacquired. Due to the rapid world population growth and other globalreasons, the study of zoonoses becomes ever more important. Recentoutbreaks of Influenza and SARS are such examples.The National Platform for Zoonoses aims to develop a network of scientiststo improve research on preparedness, prevention, detection, and control ofzoonotic diseases. Our objective is to promote exchange of expertise on thenational and international level and thus to accelerate research activities inthe field of zoonoses. In addition, we pursue the wide horizontal crosslinkingof human and veterinary medicine.These objectives will be achieved by the following activities: Organization and realization of joint events which supportinterdisciplinary exchange and interaction. Promotion of national, European and international collaborations.spektrum | Tagungsband 2011