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

SRV004A novel type of DNA photolyase containing an iron sulfurclusterI. Oberpichler* 1 , J. Wesslowski 1 , R. Pokorny 2 , R. Rosen 3 , F. Zhang 1 ,O. Neubauer 4 , A. Batschauer 2 , E. Ron 3 , T. Lamparter 11 Botany I, Karlsruhe Institute of Technology (KIT) Campus South,Karlsruhe, Germany2 Molecular Plant Physiology, Philipps-University, Marburg, Germany3 Department of Molecular Microbiology and Biotechnology, Tel AvivUniversity, Tel Aviv, Israel4 Institute for Microbiology, Humboldt-University, Berlin, GermanyPhotolyases and cryptochromes are evolutionarily related flavoproteins withdistinct functions. While photolyases can repair UV-induced pyrimidinedimers on the DNA in a light dependent manner, cryptochromes regulategrowth, development and the circadian clock in plants and animals. Here wereport about a photolyase related protein, named PhrB, found in thephytopathogen Agrobacterium tumefaciens. Phylogenetic studies showedthat PhrB belongs to a new class which we designate bacterial cryptochromeand photolyase proteins (BCP). It contain FAD as a catalytic cofactor and asecond chromophore that absorbs in the short wavelength region, but withspectral properties distinct from other known photolyase antenna cofactors.Alignment of protein sequences suggests that the classical photoreductionpathway consisting of three tryptophans, is absent in PhrB. Moreover,structure modelling revealed four cystein residues that seem to be clusteredpossibly for the coordination of an iron sulfur cluster and the presence ofiron in a 4:1 stochiometry was confirmed experimentally. Although PhrB isclearly distinguished from other photolyases it is required for photorepair ofUV-lesions in A. tumefaciens. We thus propose that PhrB is a functionalphotolyase which represents the first member of this protein family thatcontains an iron-sulfur cluster.SRV005Specific control of hypochlorite resistance by the redoxsensingMarR/DUF24-type regulator HypR in BacillussubtilisB.K. Chi 1 , G. Palm 2 , K. Gronau 1 , U. Mäder 1 , D. Becher 1 , W. Hinrichs 2 ,M. Hecker 1 , H. Antelmann* 11 Institute for Microbiology, Ernst-Moritz-Arndt-University, Greifswald,Germany2 Institute of Biochemistry, Ernst-Moritz-Arndt-University, Greifswald,GermanyBacillus subtilis encodes several redox-sensing MarR-type regulators of theOhrR and DUF24-families that are conserved among Firmicutes and controloxidative stress resistance and virulence functions in pathogenic bacteria viathiol-based redox-switches. While most characterized members of the OhrRfamily respond to organic hydroperoxides, the DUF24-family regulatorsYodB, CatR and HxlR were shown to sense specifically electrophiles suchas diamide, quinones or aldehydes. However, the genome of Bacillus subtilisencodes additional DUF24 family regulators of unknown functions and wewere interested whether any of these is involved in oxidative stressresistance mechanisms. We used DNA microarray analysis to analyseexpression changes in B. subtilis in response to the strong oxidanthypochloric acid (HOCl) which is present in house-hold bleach. The overalltranscriptional response of B. subtilis to HOCl is indicative of disulfidestress and overlapping to the response provoked by the thiol-oxidizingelectrophile diamide. The glyceraldehyde 3-phosphate dehydrogenase GapAwas most strongly oxidized to an intramolecular disulfide by HOCl stressamong cytoplasmic proteins as shown by redox proteomics and massspectrometry. We further identified an unknown DUF24-type transcriptionalregulator as novel hypochlorite-specific redox sensor which we accordinglyrenamed as HypR. HypR controls positively an oxidoreductase (HypO) thatconfers specific protection against HOCl stress in B. subtilis. The conservedN-terminal Cys residue of HypR is essentiell for activation of hypOtranscription by HOCl stress in vitro and in vivo. HypR resembles a 2-Cystype redox sensing regulator of the DUF24 family that is activated byintersubunit disulfide formation in response to HOCl stress in vitro and invivo as confirmed by mass spectrometry. Crystallization trials and structuralrefinements of oxidized and reduced HypR proteins are in progress tosupport the thiol-disulfide switch model for this novel transcriptionalactivator. Collectively our studies have revealed that the conservedMarR/DUF24 family is able to sense selectively electrophiles (diamide,quinones and aldehydes) and strong oxidants such as HOCl. Bleach is notonly present in the soil environment of B. subtilis but also released byactivated macrophages upon the infection process. Thus, the function of theDUF24 family among pathogenic Gram-positives could be to protect cellsagainst the host immune defense.SRV006Structural studies on the Iron core formation inMarinobacter hydrocarbonoclasticus DpsC. Hernandez* 1 , A. Pereira 2 , P. Tavares 2 , S. Andrade 11 Institute of Organic Chemistry and Biochemistry, Albert-Ludwigs-University, Freiburg im Breisgau, Germany2 Center of Fine Chemistry and Biotechnology, New University of Lisbon,Requimte, Monte de Caparica, PortugalIron is an essential element for the vast majority of organisms. Among othercharacteristics, its capacity to cycle between two (or more) redox states (Fe 2+or Fe 3+ ) made it an attractive element to use in the catalytic active site ofseveral enzymes [1]. In the reduced Fe 2+ ferrous state iron is relativelysoluble. In the oxidized Fe 3+ form, however, it becomes insoluble andconsequently its bioavailability in our modern oxidative atmosphere isseverely decreased [2]. Additionally, in the presence of oxygen, iron sitescan become a source of unwanted oxygen reactive species such assuperoxide or hydrogen peroxide. To overcome this problem, iron must bekept in a non-toxic reduced form, in the cell. Dps proteins (DNA-bindingprotein from starved cells), widely spread in bacteria, are highly important inthe bacterial response against oxidative stress. They are members of theferritin superfamily but, contrary to ferritins that are only involve in thebiomineralization and iron storage, Dps proteins have the capacity todetoxify the cell by removing hydrogen peroxide and ferrous iron andtherefore the ability to protect DNA against oxidative damage [3]. Dpsproteins are dodecamers with a two-fold symmetry in the dimer. Theferroxidase center lies at the interface between two monomers and has ahighly specific and conserved motif among Dps proteins; a HW pair in helixI and H-14-DXXXE in helix II where the histidines, aspartate, andglutamine residues are the iron ligands [4,5]. Despite the high conservationof these iron ligands, the occupancy of the two metal binding site differssignificantly in known crystal structures [6]. Using X-Ray crystallography incombination with spectroscopic data we are investigating the intermediatesstages of iron core formation in a Dps protein from Marinobacterhydrocarbonoclasticus.[1] Le Brun , N. E. et al (2010): Biochemica et Biophysica Acta, 1800, 732-744.[2] Bou-Abdallah , F. (2010): Biochemica et Biophysica Acta, 1800, 719-731.[3] Almiron, M. et al (1992): Genes & Development, 6, 2646-2654.[4] Andrews, S. C. (2010): Biochemica et Biophysica Acta, 1800, 691-705.[5] Ilari, A. et al. (2000): Nature Structural & Molecular Biology, 38-43.[6] Chiancone, E. & P. Ceci (2010): Biochemica et Biophysica Acta, 1800, 798-805.SRV007Characterisation of the oxidative stress response in C.glutamicumK. Marin* 1 , C. Lange 1 , C. Trötschel 2 , D. Seiferling 1 , I. Ochrombel 1 ,A. Poetsch 2 , R. Krämer 11 Institute of Biochemistry, University of Cologne, Cologne, Germany2 Plant Biochemistry, Ruhr-University, Bochum, GermanyAerobic bacteria are exposed to oxidative stress as daily problem because ofthe permanent endogenous formation of reactive oxygen species likehydrogen peroxide (H 2O 2). The cellular sources of ROS are manifold andbecause of the damage of cellular components the enzymatic removal ofROS, e.g. by catalase, plays a pivotal role in the bacterial oxidative stressresponse. Interestingly, knowledge on oxidative stress response of C.glutamicum is scarce in spite it is applied in large scale industrialfermentations and exposed to rigorous variations of the oxygen supply.Interestingly, the catalase of C. glutamicum has an extraordinary highactivity promoting its industrial production. Why does C. glutamicumpossess a highly active catalase and does the non-constitutive expression ofthe catalase gene cause metabolic limitations?We addressed the oxidative stress response of C. glutamicum and thecontribution of the catalase by comparing a catalase mutant and wild typecells. Whereas wild type cells tolerate exposure to 1 M H 2O 2 cells of themutant are highly sensitive and can not grow in presence of 1 mM H 2O 2.Additionally, an increased sensitivity towards alkaline pH and increased ironavailability was found. By using in vitro experiments the significant impactof low amounts of ferrous iron on protein oxidation was shown by theOxyblot TM technique and compared with the impact of other divalentspektrum | Tagungsband 2011