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

mineral surfaces as a biofilm (around 30 %) could be observed after 6 days.The cell density per CF volume significantly correlated with esterase activity(Pearson r = 0.77, measured with Fluorescein diacetate) or the fluorescenceintensity (Pearson r = 0.91, measured with GFP-labeled E. coli). Therespiratory activity of P. putida mainly depended on water and oxygenavailability. At high water saturation, no oxygen was available forrespiration and at low water saturation, below 7.5 % at the very top of theCF, not enough water seemed to be bioavailable. Furthermore the respiratoryactivity of cells grown on porous sand grains was always higher than of cellsgrown on smooth glass bead surfaces [2]. At sufficient nutrient supply theinterface region in a CF acts as a barrier for oxygen diffusion towards thesaturated zone: More oxygen is consumed by bacterial respiration and itsdiffusion into the water phase is limited.Our results can help to improve models for biodegradation of organicpollutants or for vertical gas transport across the CF, which presumably isinfluenced by the activity of aerobic bacteria.[1] Jost, D. et al (2010): Distribution of aerobic motile and non-motile bacteria within the capillaryfringe of silica sand. Water Research 44, 1279-1287.[2] Jost, D. et al (2011): Water and oxygen dependence of Pseudomonas putida growing in silica sandcapillary fringes. Vadose Zone Journal, in press.OTP010Will not be presented!OTP011Recombinant S-layer production induces disordered celldivision in E. coli filamentsF. Lederer*, T. Günther, J. Raff, K. PollmannInstitute of Radiochemistry, Biogeochemistry Division, Research CenterDresden-Rossendorf, Dresden, GermanyThe rod-shaped bacterium Escherichia coli is one of the best studiedmicroorganism with a size of 1.1-1.5 μm x 2.0-6.0 μm. We used E. coliBL21 (DE3), one of the most widely used host in genetic engineering, forheterologous expression of surface layer (S-layer) proteins to enable fast andefficient protein production.S-layer are proteins which cover the outermost of many prokaryotes and areprobably the basic and oldest forms of bacterial envelope. These proteins aremostly composed of protein and glycoprotein monomers and have the abilityto self-assemble into two-dimensional arrays on interfaces. Severalcharacteristics like their work as molecular sieve, as virulence factor or theprotection of the cell from toxic heavy metal ions make S-layer proteinsinteresting for their usage as ultrafiltration membranes, drugmicrocontainers, filter materials or patterning structures in nanotechnology.Surprisingly, the heterologous expression of S-layer proteins of the uraniummining waste pile isolate Lysinibacillus sphaericus JG-A12 induced drasticmorphological changes of E. coli BL21 (DE3) single cells to filaments andsingle cell enclosing tubes of >100 μm in length. The assumed secretion oftube-stabilizing S-layer proteins was investigated with SDS-PAGE and ß-galactosidase assay. These analyses result in a high S-layer appearancewithout significant ß-galactosidase activity in the supernatant and theperiplasm. The origin and composition of filaments and tubes were analysedby membrane stain studies. We identified that filaments in the exponentialgrowth phase form a continuous intracellular space without partitioning. Toinvestigate the mechanism of filament and tube formation we analyzedGFP/S-layer expressing E. coli with DAPI-stain. The staining showed >50μm long DNA-fibres that cross the filaments and „DNA-free” areas, thelatter exhibiting strong GFP-expression. Our results point to a disorderedcell division in E. coli filaments which is effected by recombinant S-layerexpression.[1] Lederer et al. (2010) Heterologous expression of the surface-layer-likeprotein SllB induces the formation of long filaments of Escherichia coliconsisting of protein-stabilized outer membrane. Microbiology 156,3584-95.OTP012Insights into the active site of the nitrogenase MoFeproteinT. Spatzal* 1 , M. Aksoyoglu 2 , S. Andrade 1 , S. Weber 2 , O. Einsle 11 Institute of Organic Chemistry and Biochemistry, Albert-Ludwigs-University, Freiburg, Germany2 Insitute of Physical Chemistry, Albert-Ludwigs-University, Freiburg,GermanyBiological nitrogen fixation is an essential process that transformsatmospheric dinitrogen (N 2) into a bioavailable form, ammonium (NH + 4 ).This process is catalyzed by the enzyme system nitrogenase, a complex oftwo metalloproteins that forms under turn-over conditions. The twocomponents of the complex are the Fe- and MoFe-proteins. The MoFeproteinfrom Azotobacter vinelandii is a 230 kDa α 2β 2-heterotetramer thatcontains two types of metal centers, the P-cluster [8Fe:7S] and the FeMocofactor[7Fe:Mo:9S:X:homocitrate] per αβ-heterodimer 1) . The FeMocofactormarks the active site of the enzyme and is the most complex metalcenter known in nature so far. Due to its complexity, the reaction mechanismis not known in detail 2) . High resolution X-ray data of the MoFe-proteinrevealed the presence of a ligand (X = C, N or O) in the center of the FeMocofactor3) which is masked by the unique metal environment in X-raystructures solved at lower (> 1.55 Å) resolutions, but which is of vitalimportance for understanding the mechanism of catalysis. Due to the limitedfeasibility of X-ray diffraction to discriminate between light atoms, acombined approach between Electron-paramagnetic-resonance (EPR) andhigh-resolution X-ray crystallography is explored. The crystallographicrefinement at < 1.1 Å as well as 12 C/ 13 C-electron nuclear resonancespectroscopy provide new insights into the nature of the cofactor and thecharacter of the central atom.[1] Hu, Y. et al (2008): Assembly of Nitrogenase MoFe Protein. Biochemistry, 47, 3973-3981.[2] Hu, Y and M. W. Ribbe (2010): Decoding the nitrogenase mechanism: the homologue approach.Acc. Chem. Res., 16, 475-484.[3] Einsle, O. et al (2002): Nitrogenase MoFe-protein at 1.16 A resolution: a central ligand in theFeMo-cofactor. Science, 297, 1696-1700.OTP013The special type IV secretion system of Neisseriagonorrhoeae: Biochemical characterization of the novelrelaxase TraI and the coupling protein TraDE.-M. Heller*, J. Koch, H.-T. Deinzer, T. Bender, S. Jain, C. van der DoesDepartment of Ecophysiology, Max Planck Institute for TerrestrialMicrobiology, Marburg, GermanyThe human pathogen Neisseria gonorrhoeae causes the sexuallytransmissible disease gonorrhoeae. Approximately 80 % of the clinicalisolates of N. gonorrhoeae contain a Gonococcal Genetic Island (GGI)which encodes a remarkable type IV secretion system (T4SS) [1, 3].However the gonococcal T4SS differs from other known T4SS in the waythat single stranded chromosomal DNA is secreted into the environment [3].The secreted DNA can be taken up via natural competence and can beintegrated into the chromosome. The high transformation frequency ofNeisseria leads to a wide spread of genetic information and results in anincrease of antibiotic resistance.T4SSs consists of a membrane spanning complex through which thesubstrates are secreted. Substrates are targeted to this complex via thecoupling protein, a hexameric ATPase. In conjugative T4SSs, thetransported DNA is initially cleaved at the oriT by the relaxase protein thatstays bound to the DNA, and is then transported to the recipient cell.Remarkably, the neisserial relaxase TraI belongs to a novel family ofrelaxases. Besides typical relaxase features this family is characterized byspecial sequence motifs: i) a conserved HD domain, ii) an alternative 3Hmotif, and iii) a C-terminal DUF1528 domain. These relaxases can be foundin Genetic Islands (GIs) as well as in conjugative plasmids and Integrativeand Conjugative Elements (ICEs) [2, 4].To date, the DNA processing mechanism and the targeting mechanism ofthis large and novel relaxase family has not been characterized. We set up abiochemical approach to characterize the relaxase TraI and the couplingprotein TraD to gain insights into the mechanism of the special T4SS of N.gonorrhoeae. Both proteins were overexpressed and purified, and here wereport a initial biochemical characterization of these proteins.[1] Dillard, J. P. and H. S. Seifert (2001): A variable genetic island specific for Neisseria gonorrhoeaeis involved in providing DNA for natural transformation and is found more often in disseminatedinfection isolates. Mol Microbiol 41(1): 263-77.spektrum | Tagungsband 2011