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

61partitioning. Out of these elements, the tubulin homologue FtsZ, the actinhomologue MreB, and intermediate filament-like (IF) proteins arewidespread in many bacterial lineages. In addition, in recent years, anincreasing number of non-canonical cytoskeletons have been identified inbacteria. These include a new class of cytoskeletal proteins, namedbactofilins, which was originally discovered in Caulobacter crescentus.Bactofilins are widely distributed among bacteria and show no similarityin either sequence or structure to other known cytoskeletal proteins.Interestingly, many species possess two or more bactofilin alleles,indicating multiple gene duplication events and functional differentiation.Previous work showed that in C. crescentus,two bactofilin paralogues,BacA and BacB, are involved in stalk biogenesis. In this study, we haveextended the investigation of bactofilins to Myxococcus xanthus, a socialspecies that contains four bactofilin homologues. Our results suggest thatbactofilins of M. xanthus are involved in a variety of different processes,mediating the proper arrangement of protein complexes within the cell.Thus, bactofilins are a novel and widespread group of cytoskeletal proteinsthat show a conserved overall architecture but have diverged significantlywith respect to their localization patterns and functions.BDP021Overexpression of Flotillins affects septum formation in BacillussubtilisB. Mielich*, J. Schneider, D. LopezResearch Center for Infectious Diseases, Institute for Molecular InfectionBiology, Würzburg, GermanyThe model organism Bacillus subtilis has been traditionally used to studythe presence of Flotillin proteins in bacteria (1-3). Flotillins are proteinsthat exclusively localize in lipid rafts of eukaryotic cells (4,5). In B.subtilis, flotillins localize in membrane microdomains that are functionallysimilar to the lipid rafts of eurkaryotes. This opens the door for usingbacteria as systems to address intrincate questions in developmentalbiology such as the role of flotillins in lipid rafts or the influence thatflotillins exerts on of diverse cellular processes that are related to lipidrafts.We constructed a strain that simultaneously overexpress the two genes thatencode for flotillin-like proteins in B. subtilis, yqfA and floT. Higherconcentration of flotillin proteins was found in the membrane of growingcells. Remarkably, the overexpression of flotillin caused hyperactivation ofseveral signaling transduction pathways associated with lipid rafts, likebiofilm formation. Moreover, overexpression of flotillins caused aberrantcell division in B. subtilis. Cells showed smaller cell size, probably causedby the assembly of multiple septa along the cells, which eventually giverise to the formation of anucleate, non-autonomous minicells that swimfreely in the cultures of B. subtilis (6). Microscopical and biochemicalstudies will be shown to elucidate how flotillin influence the properlocalization of the proteins responsible for septum formation and theactivation of the signaling pathway to biofilm formation in B. subtilis.1. C. Donovan, M. Bramkamp, Characterization and subcellular localization of a bacterial flotillinhomologue.Microbiology155, 1786 (Jun, 2009).2. D. Lopez, R. Kolter, Functional microdomains in bacterial membranes.Genes Dev24, 1893 (Sep 1, 2010).3. N. Tavernarakis, M. Driscoll, N. C. Kyrpides, The SPFH domain: implicated in regulating targeted proteinturnover in stomatins and other membrane-associated proteins.Trends Biochem Sci24, 425 (Nov, 1999).4. M. F. Langhorst, A. Reuter, C. A. Stuermer, Scaffolding microdomains and beyond: the function ofreggie/flotillin proteins.Cell Mol Life Sci62, 2228 (Oct, 2005).5. I. C. Morrow, R. G. Parton, Flotillins and the PHB domain protein family: rafts, worms andanaesthetics.Traffic6, 725 (Sep, 2005).6. H. I. Adler, W. D. Fisher, A. Cohen, A. A. Hardigree, Miniature Escherichia coli cells deficient inDNA.Proc Natl Acad Sci U S A57, 321 (Feb, 1967).BDP022Differential expression of two flotillin-like proteins in BacillussubtilisJ. Schneider*, B. Mielich, D. LopezResearch Center for Infectious Diseases, IMIB, Wuerzburg, GermanyB. subtilisis a model organism traditionally used for the study of flotillinproteins in the membrane of bacteria (1-3). Flotillins are proteinsexclusively associated with lipid rafts in eukaryotic cells (4-6). In B.subtilis and several other bacterial models, flotillin proteins localize inmembrane microdomains that are functionally similar to lipid rafts ofeukaryotic cells. Concretely, functional microdomains of B. subtiliscontain two different flotillin-like proteins named YqfA and FloT. Sincethe role of flotillins in lipid rafts is not entirely clear, we used B. subtilis asmodel organism to carry out genetic and biochemical approaches in orderto understand the role of each one of the two different flotillin-like proteinsthat are present in the functional microdomains.Our data suggest that the absence of one of the flotillins does not affect thelocalization of the other in functional membrane microdomains of B.subtilis. The expression of FloT and YqfA flotilins is controlled differentlybecause specific growing conditions lead cells to express just YqfA or bothFloT and YqfA flotillins simultaneously. Expression of YqfA orFloT+YqfA in the functional microdomains of B. subtilis affectssignificantly the functionality of the signaling pathways harbored withinthe functional microdomains. Consistently with the different expression ofthe two flotillin-like proteins, our studies of gene expression usingtranscriptional reporters indicate that floT and yqfA genes are differentlyregulated. The regulation cascades that control the expression of bothflotillin-encoding genes will be presented and discussed.1. C. Donovan, M. Bramkamp. (2009)Microbiology155, 1786.2. D. Lopez, R. Kolter. (2010)Genes Dev24, 1893.3. N. Tavernarakis, M. Driscoll, N. C. Kyrpides. (1999)Trends Biochem Sci24, 425.4. M. F. Langhorst, A. Reuter, C. A. Stuermer (2005)Cell Mol Life Sci62, 2228.5. I. C. Morrow, R. G. Parton. (2005)Traffic6, 725.6. E. Rivera-Milla, C. A. Stuermer, E. Malaga-Trillo. (2006)Cell Mol Life Sci63, 343.CEV001The bacterial MreB cytoskeleton organizes the cell membraneH. Strahl*, L. HamoenNewcastle University, Centre for Bacterial Cell Biology, Newcastle uponTyne, United KingdomMany bacteria require the actin homolog MreB to maintain a rod-like cellshape 1, 2 . This bacterial cytoskeleton protein forms short filaments beneaththe cell membrane and organizes lateral cell wall synthesis 1, 2, 3 . We foundthat compounds that perturb the localization of MreB also alter the lipiddistribution in the cell membrane. Importantly, this effect leads to anaberrant distribution of membrane proteins. We show for the E. coli LacYpermease and F 1F o ATP synthase that this is accompanied by a reductionin enzyme activity. It appears that the MreB cytoskeleton, together withthe transmembrane proteins MreC and MreD, actively organize thebacterial cytoplasmic membrane by forming fluid membrane microdomains.This property is comparable to that described for the eukaryoticcortical actin cytoskeleton 4, 5 . We speculate that this common function ofMreB and actin might be the reason why this protein family has remainedconserved during evolution.1: Graumann PL (2007) Cytoskeletal elements in bacteria. Annu Rev Microbiol 61:589-618.2: Carballido-Lopez R (2006) The bacterial actin-like cytoskeleton. Microbiol Mol Biol Rev 70(4):888-909.3: Dominguez-Escobar J, et al. (2011) Processive movement of MreB-associated cell wallbiosynthetic complexes in bacteria. Science 333(6039):225-228.4: Liu AP & Fletcher DA (2006) Actin polymerization serves as a membrane domain switch inmodel lipid bilayers. Biophysical Journal 91(11):4064-4070.5: Petrov EP, Ehrig J, & Schwille P (2011) Near-critical fluctuations and cytoskeleton-assistedphase separation lead to subdiffusion in cell membranes. Biophysical Journal 100(1):80-89.CEV002Cell envelope stress response in cell wall-deficient L-forms ofBacillus subtilisD. Wolf* 1 , P. Domínguez-Cuevas 2 , R. Daniel 2 , T. Mascher 11 LMU München, Department I, Mikrobiologie, Martinsried, Germany2 University of Newcastle upon Tyne, CBCB, Newcastle upon Tyne, UnitedKingdomL-forms are cell wall-deficient cells that can grow and proliferate inosmotically stabilizing media [1]. Recently, a strain of the Gram-positivemodel bacterium Bacillus subtilis was constructed that allows a controlledswitching between rod-shaped wild type cells and corresponding L-forms[2]. Both states can be stably maintained under suitable culture conditions.Because of the absence of a cell wall, L-forms are known to be insensitiveto -lactam antibiotics. But reports on the susceptibility of L-forms toother antibiotics that interfere with membrane-anchored steps of cell wallbiosynthesis are sparse, conflicting and strongly influenced by strainbackground and method of L-form generation. We therefore aimed atinvestigating the response of B. subtilis to the presence of cell envelopeantibiotics, both with regard to antibiotic resistance and the induction ofthe known LiaRS- and BceRS-dependent cell envelope stress biosensors[3]. Our results show that B. subtilis L-forms are resistant to antibioticsthat interfere with the bactoprenol cycle, such as bacitracin andvancomycin, but are hyper-sensitive to nisin and daptomycin, which bothaffect membrane integrity. Moreover, we established a lacZ-based reportergene assay for L-forms and provide evidence that LiaRS senses itsinducers indirectly (“damage-sensing”), while the Bce module presumablydetects its inducers directly (“drug-sensing”) [4].[1] Onoda et al. (1992), J Gen Microbiol. 138:1265-70[2] Leaver et al. (2009), Nature. 457:849-53[3] Jordan et al. (2008), FEMS Microbiol. Rev. 32:107-146[4] Wolf et al. (2011), submittedCEV003Mechanism of substrate recognition of the tRNA-dependent alanylphosphatidylglycerolsynthase from Pseudomonas aeruginosaS. Hebecker* 1 , W. Arendt 1 , T. Hasenkampf 1 , I. Heinemann 2 , D. Söll 2 ,D. Jahn 1 , J. Moser 11 TU Braunschweig, Department of Microbiology, Braunschweig, Germany2 Yale University, Department of Molecular Biophysics and Biochemistry,New Haven, United StatesThe alanyl-phosphatidylglycerol synthase (A-PGS) from the opportunisticbacterium Pseudomonas aeruginosa catalyzes the alanylation of thephospholipid phosphatidylglycerol in a tRNA Ala -dependent reaction. Whenexposed to acidic growth conditions, P. aeruginosa synthesizes significantamounts of alanyl-phosphatidylglycerol (A-PG). Furthermore, formationBIOspektrum | Tagungsband 2012