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

58Here, multiple parameters were analyzed in single cells ofCorynebacterium glutamicum via FACS. By analyzing a typical growthcurve of C. glutamicum subpopulations were identified differing in size,DNA pattern, metabolic activity, membrane integrity, and membranepotential. These populations show a dynamic pattern depending on strainbackground, cultivation conditions, and growth phase. DNA patternsoscillate within the growth curve. Cells in the early log phase containmainly a single chromosome equivalent followed by a proliferation phasecharacterized by a decrease in cells with a single chromosome equivalentand an increase in cells with multiple chromosome equivalents. Cells inthe stationary phase exhibit predominantly a single chromosomeequivalent. The reductase activity (indicator for electron transport chainfunction and cellular viability) also shows significant correlation with celldensities. Cells of the log phase show the highest reductase activitywhereas cells of the early or late log phase exhibit a reduction in viability;stationary cells demonstrate the lowest activity as well as a depolarizationof the cell membrane which could not be detected in cells of the log phase.The rate of depolarization correlates with the uptake of propidium iodideindicative for damaged cell membranes. These results demonstrate flowcytometry as an efficient tool for the study of bacterial populationdynamics and process monitoring at single cell resolution.BDP008Polar magneto-aerotaxis in Magnetospirillum gryphiswaldenseF. Popp* 1 , J. Hofmann 1 , D. Bartolo 2 , D. Schüler 11 LMU München, Mikrobiologie, AG Schüler, Planegg-Martinsried, Germany2 ESPCI-ParisTech, PMMH Lab, Paris, FranceThe paradigmatic concept of random walk motion observed in mostprokaryotes is greatly simplified in freely swimming magnetotacticbacteria (MTB) which contain a chain of nano-sized magnetic particles.Passive alignment with the Earth’s magnetic field forces the bacteria ontoa nearly linear track. In addition, most MTB possess the selectable trait tofollow the magnetic field lines in a preferred swimming direction (eitherN- or S-seeking) depending on the prevailing habitat conditions. To date,the underlying molecular mechanism of how magnetic polarity isintegrated with other taxis mechanisms is not understood.M. gryphiswaldense is a bipolarly flagellated gradient organism which iscapable of polar swimming behaviour if grown under selective conditions.Automated video tracking of wild type cells revealed swimming episodesin alternating directions which are interrupted by short reversals. Thereversal frequency did not change significantly in polarised cultures.We identified four chemotaxis gene clusters containing conserved genescheAWYBR in the genome of M. gryphiswaldense. Whereas deletion ofoperons 2-4 did not impact on chemotaxis, only loss of CheOp1 had a cleareffect on aerotaxis. This indicated a possible link between polarity andchemotaxis at the genetic level. In magnetospirillum cells having an apparentlysymmetrical morphology, polarity might be established by asymmetriclocalization of constituents of the chemotaxis machinery. Therefore, we studiedthe intracellular localization of fluorescent protein fusions to chemotaxisproteins CheA and CheW. Since these proteins localised to variable positions inthe cell they are unlikely to determine polarity.Swimming polarity is currently being studied quantitatively by amicrofluidic assay using fluorescence-labelled cells and in competitionassays. This will also reveal the putative selective advantage ofmagnetotaxis.BDP009Spatiotemporal patterns of microbial communities in ahydrologically dynamic alpine porous aquifer (Mittenwald,Germany)Y. Zhou*, C. Kellermann, C. GrieblerHelmholtz Zentrum München, Institute of Groundwater Ecology, Munich,GermanyIt has been repeatedly shown for aquatic habitats that microbialcommunities underlie seasonal dynamics and follow environmentalgradients. Recently, microbial communities of karst aquifers were shownto be influenced by seasonal hydrodynamics. In turn, we investigatedseasonal patterns of selected microbial and physical-chemical variables ingroundwater and sediments of an alpine oligotrophic porous aquifer over aperiod of two years. Characterized by a high hydraulic conductivity andgroundwater flow velocities, this aquifer exhibited pronounced seasonalhydrological dynamics, which are confirmed by pronounced groundwatertable fluctuations. The groundwater table was found highest duringsummer along with lowest bacterial diversity (H’ = 1.31 ± 0.35 SD) insuspended bacterial communities, as analyzed by T-RFLP fingerprinting.A similar pattern was observed for the total number of planktonic bacteria,with lowest numbers in spring and summer (1.4×10 4 cells mL -1 ) andhighest values (2.7 ×10 5 cells mL -1 ) during winter season. The ratio of totalversus active cells, determined by analysis of intracellular ATP, waslowest in summer and winter (0.07%~10%) and highest in autumn(16%~85%). Bacterial carbon production measurements revealed highestactivities in summer and lowest in winter, with average carbon productionof 6.22 and 1.30 ng C L -1 h -1 respectively. The carbon turnover related toconcentrations of AOC, which ranged from 5 to 25 g L -1 , accounting foronly 0.1 to 1.3% of the bulk DOC. Sediment bacterial communities from anearby river exhibited a stable community composition and diversity whenexposed to groundwater for one year. Initially sterile sediments, on theother hand, were readily colonized and established a bacterial diversitysimilar to the exposed river sediment. In conclusion, hydrodynamicsmarkedly influenced the planktonic bacterial communities while attachedcommunities have not been affected by the serious hydrological changes.BDP010During stress Spx hits the emergency brake on swimming motilityN. Moliere*, K. TurgayLeibniz Universität, Institut für Mikrobiologie, Hannover, GermanyClp proteases are key players in the regulation of bacterial differentiationprocesses, such as competence [4] and sporulation [3]. The proteaseClpXP is required for differentiation into motile cells in Bacillus subtilis[1]. An important proteolysis substrate of ClpXP is the globaltranscriptional regulator Spx, which activates the expression of stresstolerance genes during oxidative stress. At the same time Spx acts as anegative regulator of a distinct group of genes, including those responsiblefor competence development. Under non-stress conditions, Spx isefficiently degraded by the ClpXP protease, resulting in a low steady statelevel of the protein. However, in response to oxidative stress, thisproteolysis is halted and Spx accumulates in the cell [2]. We haveinvestigated the effect of ClpXP on swimming motility and found that theClpXP substrate Spx acts as a negative regulator of flagellar genes.Furthermore, motility genes are transiently down-regulated in response tooxidative stress. We propose that during adverse environmental conditions,such as oxidative stress, the execution of the stress response program isgiven priority over motility development by the action of the regulatorSpx. Possible mechanisms of this negative regulation are discussed.[1] Msadek T, Dartois V, Kunst F, Herbaud ML, Denizot F, Rapoport G.ClpP of Bacillus subtilis isrequired for competence development, motility, degradative enzyme synthesis, growth at hightemperature and sporulation. Mol Microbiol. 27(5):899-914; 1998.[2] Nakano S, Zheng G, Nakano MM, Zuber P. Multiple pathways of Spx (YjbD) proteolysis inBacillus subtilis. J Bacteriol. 184(13):3664-70; 2002.[3] Pan Q, Garsin DA, Losick R. Self-reinforcing activation of a cell-specific transcription factor byproteolysis of an anti-sigma factor in B. subtilis. Mol Cell. Oct;8(4):873-83; 2001.[4] Turgay K, Hahn J, Burghoorn J, Dubnau D. Competence in Bacillus subtilis is controlled byregulated proteolysis of a transcription factor. EMBO J. Nov 16;17(22):6730-8; 1998.BDP011Subcellular compartmentalization of a bacterial organelle byprotein diffusion barriersS. Schlimpert* 1,2 , A. Briegel 3 , K. Bolte 2 , J. Kahnt 4 , U.G. Maier 2 ,G.J. Jensen 3 , M. Thanbichler 1,21 Max Planck Institue for Terrestrial Microbiology, Max Planck ResearchGroup “Prokaryotic Cell Biology”, Marburg, Germany2 Philipps University, Department of Biology, Marburg, Germany, Germany3 California Institute of Technology, Division of Biology and HowardHughes Medical Institute, Pasadena, CA, United States4 Max Planck Institute for Terrestrial Microbiology, Department ofEcophysiology, Marburg, GermanyIntracellular compartmentalization by different diffusion barriermechanisms has previously been thought to be solely utilized byeukaryotic cells. Here, we report for the first time that non-membranousprotein diffusion barriers also exist in prokaryotes. Using Caulobactercrescentus as a model organism, we show that these diffusion barriersphysically separate cell envelope components of the cell body from thethin stalk appendage and create intra-stalk domains. The Caulobacter stalkrepresents a thin extension of the cell envelope that is free of DNA,ribosomes and most cytoplasmic proteins. It is segmented at irregularintervals by so-called crossbands, disk-like structures of so far unclearfunction and identity.In this study, we discovered that crossbands serve as protein diffusionbarriers. The major constituents of these diffusion barriers are four proteinsthat co-assemble in a cell cycle-dependent manner into a static complex atthe junction between the stalk and the cell body. Using fluorescence loss inphotobleaching (FLIP) we observed that, in contrast to eukaryotic cells,these diffusion barriers not only laterally compartmentalize cellularmembranes but also limit the free diffusion of soluble (periplasmic)proteins. Moreover, competition assays with wild-type and barrierdeficientcells revealed that diffusion barriers are essential for fitness asthey minimize the effective volume of the cell body envelope, therebyallowing faster adaptation to environmental changes that require theupregulation of protein production.Collectively, our findings demonstrate that crossband formation in thestalked alpha-proteobacterium Caulobacter crescentus presents a novelmechanism to optimize growth by restricting protein mobility in aprokaryotic cell.BIOspektrum | Tagungsband 2012