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

192network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart fromCys49'. HypR oxidation breaks these H-bonds, reorients the monomersand moves the major groove recognition alpha4 and alpha4' helices ~4 Åtowards each other. This is the first crystal structure of a redox-sensingMarR/DUF24 family protein in bacteria that is activated by NaOCl stress.Since hypochloric acid is released by activated macrophages as majordefense mechanism, related HypR-like regulators could function to protectpathogens against the host immune defense.[1] Antelmann, H., and Helmann, J.D. (2011) Thiol-based redox switches and gene regulation.Antioxid Redox Signal. 14: 1049-1063. Review.[2] Palm, G., Chi, B.K., Waack, P., Gronau, K., Becher, D., Albrecht, D., Hinrichs, W., Read, R.J.and Antelmann, H. Structural Insights into the redox-switch mechanism of the MarR/DUF24 familyregulator HypR. Nucleic Acid Research, In Revision.RSV004How the P II protein from Synechococcus integrates metabolicwith energy signals to control its targets.O. Fokina*, K. ForchhammerUniversity Tübingen, Organismic Interactions, Tübingen, GermanyP II signal transduction proteins have key functions in coordination ofcentral metabolism by integrating signals from carbon, nitrogen andenergy status of the cell. They bind the metabolites ATP, ADP and 2-oxoglutarate (2-OG) and control enzymes, transporters and transcriptionfactors involved in nitrogen metabolism. Depending on its effectormolecule binding status, P II from Synechococcus elongatus binds a smallprotein termed PipX, which is a co-activator of the transcription factorNtcA, and regulates the key enzyme of the cyclic ornithine pathway, N-acetyl-L-glutamate kinase (NAGK). P II binds ATP and 2-OG in asynergistic manner, with the ATP-binding sites also accepting ADP.Different ADP/ATP ratios strongly affect the properties of P II signalingincluding 2-OG binding and interactions with its target proteins. ADPmodulates P II signaling to the receptor NAGK primarily at low 2-OGlevels and antagonises the inhibitory effect of 2-OG for P II-PipXinteraction. Apparently P II has a fine-tuned mechanism of sensing bothchanging energy charge and carbon/nitrogen balance at the same time.Fokina O., Chellamuthu VR., Zeth K., & Forchhammer K. (2010) A novel signal transduction protein PIIvariant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-PII complexformation. J. Mol. Biol. 399:410-421.Fokina O., Herrmann C. & Forchhammer K. (2011) Signal transduction protein PII from Synechococcuselongatus PCC 7942 senses low adenylate energy charge in vitro. Biochem. J. 440:147-56.RSV005Post-translational modification determines the substratespecificity of a carboxylic acid-coenzyme A ligaseJ. Oberender*, M. BollUni Leipzig, Biochemie, Leipzig, GermanyIn anaerobic bacteria most aromatic growth substrates are converted to thecentral intermediate benzoyl-CoA. E.g., in case of benzoate and p-hydroxybenzoate degradation, the initial steps are usually catalyzed byindividual, highly specific carboxylic acid CoA ligases 1 . We established afirst genetic system for the obligately anaerobic model organismGeobacter metallireducens and disrupted the gene encoding benzoate-CoAligase (bamY) as a proof of principle. This enzyme is highly specific forbenzoate as substrate 2 . Unexpectedly, a bamY - mutant was still able togrow on benzoate with a growth rate similar to that of the wild type. Inagreement, we identified a previously unknown succinyl-CoA:benzoateCoA transferase, which obviously fully compensated for the bamYknockout. Surprisingly, the bamY - mutant was no longer able to utilize p-hydroxybenzoate as carbon source, although isolated BamY was unable toactivate p-hydroxybenzoate. Growth on p-hydroxybenzoate was observedagain in the presence of a plasmid expressing intact bamY, suggesting thatbamY is involved in p-hydroxybenzoate catabolism. Strictly dependent onacetyl-CoA, incubation of purified BamY with dialyzed extract of cellsgrown on p-hydroxybenzoate converted BamY to a p-hydroxybenzoate-CoA ligase. Results of MS-analysis of tryptic digests suggest that differentpatterns of N -lysine acetylation were responsible for altered substratespecificities of BamY. Though N -acetylation of active site lysines hasbeen reported to switch the activity of carboxylic acid CoA ligases off 3 , themodulation of substrate specificity via post-translational N -lysineacetylation was previously known.1 Fuchs G (2008), Ann N Y Acad Sci 1125:82-992 Wischgoll et al. (2005), Mol Microbiol 58:1238-12523 Crosby et al. (2010), Mol Microbiol 76:874-888RSV006Hot signal transduction in the thermoacidophiliccreanarchaeum Sulfolobus acidocaldariusD. Esser* 1 , J. Reimann 2 , T.K. Pham 3 , S.V. Albers 2 , P.C. Wright 2 , B. Siebers 1,21 Universitiy of Duisburg-Essen, Molecular Enzyme Technology &Biochemistry, Essen, Germany2 Molecular Biology of Archaea, Max Planck Institute for TerrestrialMicrobiology, Marburg, Germany3 ChELSI Institute, Department of Chemical and Biological Engineeri, Sheffield,United KingdomPosttranslational modifications are of major interest for the regulation ofcellular processes. Reversible protein phosphorylation is the mainmechanism to control the functional properties of proteins in response toenvironmental stimuli [1]. In the 80’s protein phosphorylation has beendemonstrated in the third domain of life, the Archaea [2]. However, so faronly few phospho(p) proteins were identified and few protein kinases andprotein phosphatases were investigated. The archaeal phosphorylationmachinery in general resembles more the eucaryal (Ser, Thr and Tyrphosphorylation) than the bacterial machinery (two- and one-componentsystems, Asp and His phosphorylation). Bioinformatic analysis revealedthat Ser, Thr and Tyr phosphorylation is ubiquitous in Archaea, whereastwo- and one-component systems are only present in the euryarchaeota(e.g. CheA/CheY in Halobacterium salinarium) [1,3].Model organism of this study is the thermoacidophilic CreanarchaeonSulfolobus acidocaldarius, with optimal growth at 78°C and pH of 2-3 [4].Bioinformatic investigation revealed that S.acidocaldarius harbors twelveprotein kinases and two protein phosphatases [1]. Nine of the twelveidentified protein kinases (PK) show high sequence similarity to eukaryaltype like protein kinases and the remaining three to atypical proteinkinases. The two protein phosphatases (PP) show similarity to proteintyrosine phosphates (PTP) and protein phosphatases (PPP). Furthermore,Sulfolobus species itself have an unusual high PK to PP ratio (12:2)compared to other archaea (3:1 to 1:1) [1]. First analysis of the p-proteomerevealed a high no. of p-proteins and a high no. of p-Tyr (Ser 31.8%, Thr24.8%, Tyr 43.3%). The detected p-proteins are found in all major arCOGcategories.In order to investigate signal transduction in S. acidocaldarius we clonedand characterized the PP2A catalytic subunit from S. acidocaldarius. Untilknow, all investigated archaeal PPPs are members of the PP1-arch [5-7]and so far no member of the PP2-arch was characterized. This is the firstdetailed characterization of an archaeal PP2A. The current understandingof signal transduction in S. acidocaldarius with focus on the PP2A will bepresented.1. Kennelly, P.J., Biochemical Journal, 2003. 370(2): p. 373-389.2. Spudich, J.L. and W. Stoeckenius, Journal of Biological Chemistry, 1980. 255(12): p. 5501-5503.3. Rudolph, J. and D. Oesterhelt, EMBO Journal, 1995. 14(4): p. 667-673.4. Grogan, D.W., Journal of Bacteriology, 1989. 171(12): p. 6710-6719.5. Mai, B., et al., Journal of Bacteriology, 1998. 180(16): p. 4030-4035.6. Solow, B., J.C. Young, and P.J. Kennelly, Journal of Bacteriology, 1997. 179(16): p. 5072-5075.7. Leng, J., et al., Journal of Bacteriology, 1995. 177(22): p. 6510-6517.RSV007A novel LuxR-based cell-to-cell communication system in theentomopathogen Photorhabdus luminescensI. Hitkova 1 , C. Manske 1 , S. Brameyer 1 , K. Schubert 1 , C. Harmath 1 ,S. Linnerbauer 1 , S. Joyce 2 , D. Clarke 2 , R. Heermann* 11 Ludwig-Maximilians-Universität München, Biozentrum, BereichMikrobiologie, Martinsried/München, Germany2 University College Cork, Department of Microbiology and AlimentaryPharmabiotic Centre, Cork, Ireland, IrelandCell-to-cell communication via acyl-homoserine lactones (AHL) is wellstudied in many Gram-negative bacteria. The prototypical communicationsystem consists of a LuxI-type autoinducer synthase and a LuxR-typereceptor that detects the endogenously produced signal. The symbiotic andentomopathogenic enteric bacterium Photorhabdus luminescens harbors39 LuxR-like receptors, but lacks any LuxI-type autoinducer synthase andis unable to produce AHL. It is unclear whether P. luminescens uses theseorphan LuxR homologues for the detection of exogenous or endogenoussignals. In this study we demonstrate that P. luminescens does engage inendogenous LuxR-based cell-cell communication. We show that one of theLuxR homologues, Plu4562 (PluR), detects an endogenously producedsignaling molecule (PLAI-1) that is not an AHL but, rather, a 2-pyronederivative named photopyrone. We also show that PluR positivelyregulates the expression of the plu4568-plu4563 operon, encoding genesinvolved in cell clumping. However plu4568-plu4563 is not responsiblefor the production of PLAI-1 and the nature of the clumping factorproduced by this operon remains unidentified. We also show that theLysR-type regulator HexA, which is a global repressor of symbiosis genesin P. luminescens, represses plu4568-plu4563 expression. This suggeststhat the plu4568-plu4563 operon may be involved in the mutualisticinteraction between P. luminescens and the nematode. Indeed we haveshown that colonization of the symbiotic partner Heterorhabditisbacteriophora by a P. luminescens pluR mutant does appear to beBIOspektrum | Tagungsband 2012