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

[1] Fokina, O. et al (2010): A Novel Signal Transduction Protein P(II) Variant from Synechococcuselongatus PCC 7942 Indicates a Two-Step Process for NAGK-P(II) Complex Formation. J Mol Biol399:410-421.[2] Fokina, O. et al (2010): Mechanism of 2-Oxoglutarate signalling by the Synechococcus elongatusPII signal transduction protein. Proc Natl Acad Sci USA 107:19760-19765.PSV001Do Gram positives recycle their cell wall?C. MayerDepartment of Molecular Microbiology, University of Konstanz, Konstanz,GermanyThe peptidoglycan, the stabilizing component of the bacterial cell wall, isnot inert but is permanently degraded, remodelled, and re-synthesized duringcell growth and differentiation. Although the release of a substantial amountof peptidoglycan turnover products (muropeptides) has been reported formany bacteria, their reutilization (cell wall recycling) has been studied, sofar, only in the Gram-negative bacterium Escherichia coli. The Grampositivecell wall differs from the Gram-negative cell envelope by the lackof an outer membrane, by the formation of a thick, multi-layeredpeptidoglycan that contrasts to the essentially single-layered peptidoglycanof Gram-negative bacteria, and by the presence of long anionic polymerscalled teichoic acids that are covalently attached to the peptidoglycan (wallteichoic acids). Therefore, cell wall turnover in Gram-positive bacteria hasto proceed different from the Gram-negative pathway. Whether the cell wallturnover products in Gram-positives are also recycled and under whichconditions this may occur is currently unclear. We identified pathways thatare used for the recovery of N-acetyl-glucosamine (GlcNAc)-N-acetylmuramicacid (MurNAc)-peptides (muropeptides) derived from the cell wallin Bacillus subtilis and Clostridium acetobutylicum. Interestingly, mutationswithin this pathways result in lytic phenotypes. We explored the conditionsfor autolysis, cell wall shedding and recovery in these Gram-positivebacteria and characterized the enzymes of these pathways.PSV002A RubisCO-like Protein links SAM-Metabolism withIsoprenoid BiosynthesisT.J. Erb*, J.A. GerltInstitute for Genomic Biology, University of Illinois at Urbana-Champaign,Urbana, IL, USAD-Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is one ofthe most abundant enzymes in the biosphere and catalyzes the key reactionof the Calvin cycle, the major process of CO 2-fixation on earth. To date,three different types of RubisCO have been identified that all serve as truecarboxylases in plants, bacteria, and archaea, respectively [1].Recent sequencing projects identified close RubisCO-homologues(RubisCO-like proteins) in a number of bacterial and archaeal genomes,such as Bacillus subtilis, Pseudomonas putida, Mesorhizobium loti,Chlorobaculum tepidum, Archaeoglobus fulgidus, or Rhodospirillumrubrum. In contrast to true RubisCOs, these RubisCO-like proteins (RLPs)miss residues essential for the carboxylation reaction and consequently lackthe ability to fix CO 2. However, genomic context and active site residuedifferences suggest that all these RLPs serve different physiologicalfunctions.We recently assigned a function to the RubisCO-like protein ofRhodospirillum rubrum, studying its mechanistic diversity in vitro. ThisRLP can use methylthioribulose-1-phosphate as substrate to catalyze twosubsequent enolization reactions [2]. Further investigation on thephysiological significance of this new reaction in vivo was carried out usinga combined approach of RNA sequencing (RNAseq), knockoutmetabolomics, cell extract NMR, and functional enzymology.Our results led to the identification of a completely novel bacterial strategyto salvage methylthioadenosine, a dead end product of S-adenosylmethionine (SAM) in spermidine and biotin biosynthesis. This strategyinvolves the release of methanethiol (CH 3SH) from the carbon skeleton,whereas the rest of the molecule is transformed into deoxyxylulose-5-phosphate (DXP), an essential intermediate in isoprenoid biosynthesis.In summary, the RubisCO-like protein of R. rubrum provides a novelbiosynthetic route to isoprenoids by linking two key processes of purplenon-sulfur bacteria, polyamine and carotenoid biosynthesis, in an efficientand elegant manner. These findings will add another piece to ourunderstanding of the evolutionary and functional relationship betweenRubisCO and RubisCO-like proteins.[1] Tabita, F.R. et al (2007): Microbiol Mol Biol Rev. 71:576-99.[2] Imker, H.J. et al (2008) Biochemistry. 47:11171-3.PSV003Flagellar motor tuning - a novel hybrid motor inShewanella oneidensis MR-1A. Paulick*, K. ThormannDepartment of Ecophysiology, Max Planck Institute Marburg, Marburg,GermanyThe flagellar motor consists of two major structures: the rotor, which is therotating component and the stator, which provides a fixed component in themembrane. The stator complexes are thought to surround the rotor, however,the stator ring system is surprisingly dynamic. It has recently been shownthat stator complexes are constantly exchanged with a membrane locatedpool of precomplexes which are activated upon incorporation into the motor.Our physiological and localization studies on Shewanella oneidensis MR-1revealed that two different sets of stators, annotated as PomAB (sodium iondependent)and MotAB (proton-dependent) differentially support theflagellar rotation. Our current working model suggests that PomAB andMotAB are present as precomplexes in the cell membrane and compete forincorporation into the stator ring system. High sodium ion concentrationsstrongly favour incorporation of PomAB stator complexes, whereas lowsodium ion concentrations decrease the presence of PomAB statorcomplexes. Instead the proton-driven MotAB stator complexes are recruited.Our data strongly suggest that under low sodium ion concentrations theflagellar motor is simultaneously driven by PomAB and MotAB statorcomplexes. We therefore propose that the single polar flagellum of S.oneidensis MR-1 is powered by a hybrid motor which concurrently usessodium ions and protons. Interestingly, our in silico analysis of 400organisms with a single flagella system revealed that 134 organisms harbormultiple stator complexes. Thus, adaptation to different environmentalconditions might be conferred by stator swapping.So far, the natural occurrence of a hybrid motor has never beendemonstrated. However, our data provide strong indications that S.oneidensis MR-1 harbors this novel kind of a hybrid motor to adapt toenvironmental changes. In addition, we propose that stator swapping tomodify motor functions is widespread among bacteria.PSP001Nutrient depending volatile emission of Serratia odorifera4Rx13T. Weise*, M. Kai, B. PiechullaInstitute of Biological Sciences, Biochemistry, University of Rostock,Rostock, GermanyThe Gram-negative rhizobacterium Serratia odorifera 4Rx13 emits a wealthof volatiles. Such volatiles possess different effects on neighboringorganisms plants, fungi, protozoa [1]. Within the volatile mixture S.odorifera emits a major compound, with a structure new to science.(Octamethylbicyclo(3.2.1)octadiene, `Sodorifen´) [2,3]. The underlyingbiosynthesis of this compound is completely unknown. The unusual massspectrum is accompanied by several isomers which indicate along with C 13labelled acetate experiments a novel pathway of `sodorifen´. Two strategiesare presently pursued to unravel the biosynthesis and regulation of this newcompound, i) genetic analysis and ii) physiological analysis. The latterincludes tests on various media such as complex media, +/- glucose, orsynthetic media +/- amino acids or +/- variety of carbon sources. Highest‘sodorifen’ production was observed on complex medium or on syntheticmedium with the addition of three amino acids. Furthermore, experimentswith C 13 labelled methionine advert that only one of the eight methyl groupsoriginates from a methyltransferase reaction. The genetic analysis becamepossible after sequencing the full genome of S. odorifera (NCBI Project ID42253). Currently a knock out system will be established to allow the test ofcandidate genes involved in the biosynthesis of `sodorifen´.Acknowledgement: We thank our collaborators W. Francke, S. von Reuß(University of Hamburg, D), G. Gottschalk, R. Daniel, A. Thürmer, J. Voss,R. Lehmann (University of Göttingen, D) and E.Crespo, S. Cristescu, F.vHarren (Nijmegen, NL).[1] Wenke, K. et al (2010): Planta 231: 499-506.[2] Kai, M. et al (2010): AMB 88: 965-976.[3] Von Reuß, S.et al (2009): Angewandte Chemie 122:2053-2054.spektrum | Tagungsband 2011