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

NTP003Resolution of natural microbial community dynamics bycommunity fingerprinting, flow cytometry and trendinterpretation analysisS. Müller, S. Kleinsteuber*, P. Bombach, I. Fetzer, T. HübschmannDepartment of Environmental Microbiology, Helmholtz Center forEnvironmental Research (USZ), Leipzig, GermanyNatural microbial communities have generally an unknown structure andcomposition due to their still not yet cultivable members. Therefore,understanding the relationships between the bacterial members, prediction oftheir behaviour and controlling their functions is a difficult and often onlypartly successful endeavour to date. This study aims to test a new idea whichallows following community dynamics on the basis of a simple concept.Terminal restriction fragment length polymorphisms (T-RFLP) analysis ofbacterial 16S ribosomal RNA genes was used to describe a communityprofile which we define as composition of a community. Flow cytometryand analysis of DNA contents and forward scatter characteristics of thesingle cells were used to describe a community profile which we define asstructure of a community. Both approaches were brought together by a nonmetricmultidimensional scaling for trend interpretation of changes in thecomplex community data sets. This was done on the basis of a graphicalevaluation of the cytometric data, leading to the newly developedDalmatian-plot tool, which gave an unexpected insight into the dynamics ofthe unknown bacterial members of the investigated natural microbialcommunity. The approach presented here was compared with othertechniques described in the literature.The microbial community investigated in this study was obtained from aBTEX contaminated anoxic aquifer. The indigenous bacteria were allowedto colonize in in situ microcosms consisting of activated carbon. Thesemicrocosms were amended with benzene and either of the electron acceptorsnitrate, sulfate or ferric iron to stimulate microbial growth. The dataobtained in this study indicated that the composition (via T-RFLP) andstructure (via flow cytometry) of the natural bacterial community wereinfluenced by the hydro-geochemical conditions in the test site but also bythe supplied electron acceptors which led to distinct shifts in relativeabundances of specific community members.It was concluded that engineered environments can be successfullymonitored by single cell analytics in combination with established moleculartools and sophisticated statistical analyses, a mélange, which holds greatpromise for studying and monitoring natural microbial communitybehaviour.NTP004Phosphorylation mechanisms of bacterial organismsshow the importance of energy thresholds in living RNAsystems.S. Lawrence*, S. LawrenceEarth Sciences and Biochemistry Research, University of Cambridge,Cambridge, United KingdomThe mechanisms of phosphorylation is of vital importance for the livingorganism, whether at bacterial level or at eukaroytic level. The processoccurs in all RNA processes and the actual atomic level process ofphosphorylation and the energy requirements for this process provide a largeinsight into how the living system uses natural energy in cell processes.Thedetailed energy levels can be calculated,and as will be shown in thepresentation,can then be manipulated for comparisons with otherintracellular processes and even to extracellular processes.This means thatthere is a standard to which future phosphorylation mechanisms in RNA canbe measured. The detailed method of this will be shown and photographicrepresentations of RNA processes related to phosphorylation will be shownin the presentation.Photographic representations are useful in visualisinghow the phosphorylation process is progressing in different parts of theRNA activities.NTP005Measuring unbiased metatranscriptomics in pelagicaerobic ammonium oxidation zones of the central BalticSeaM. Labrenz* 1 , J. Feike 1 , J.T. Hollibaugh 2 , S. Krüger 1 , G. Jost 1 , K. Jürgens 11 IOW-Leibniz Institute for Baltic Sea Research, Biological Oceanography,Rostock-Warnemuende, Germany2 University of Georgia, Department of Marine Sciences, Athens, USAMicroorganisms mediate all geochemical cycles relevant to sustaining lifeon Earth. An analysis of their metabolism is therefore fundamental tounderstanding globally important element transformations. However, mostmicrobes are recalcitrant to cultivation, such that culture-independentmethods must be used to deduce their metabolic functions. One approachthat has already shown great promise in this regard is to analyze the pool oftranscripts contained in natural microbial assemblages (metatranscriptomes).Unfortunately, since mRNA is extremely labile and can degrade in less than30 sec, it is unclear whether the abundance patterns detected in nature arevulnerable to considerable modification in situ simply due to samplingprocedures. Exemplified on comparisons of metatranscriptomes retrievedfrom pelagic aerobic ammonium oxidation zones the central Baltic Sea (70-120 m depth) and quantification of the specific transcripts in them, we showthat different sampling techniques significantly influence the relativeabundance of transcripts presumably diagnostic of the habitat. In situfixation using our newly developed automatic flow injection samplerresulted in an abundance of crenarchaeal ammonia monooxygenasetranscripts that was up to 30-fold higher than that detected in samplesobtained using standard oceanographic systems. By contrast, the abundanceof transcripts indicative of cellular stress was significantly greater in nonfixedsamples. Thus, the importance of in situ fixation in the reliableevaluation of distinct microbial activities in the ecosystem based onmetatranscriptomics is obvious. Taken these results, this could also be thecase in attempts aimed at an unbiased analysis of areas below the epipelagiczone, which cover 90% of the world's oceans.NTP006Will be presented as oral presentation with the ID NTV005!NTP007Live / dead discrimination of biofilm bacteria from adrinking water pilot distribution systemJ. Varela Villarreal*, C. Jungfer, U. Obst, T. SchwartzInstitute of Functional Interfaces, D epartment of Interface Microbiology,Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen,GermanyFormation of biofilms in drinking water distribution networks, includingpipelines of households and food industries, are of great concern. Biofilmsare potential habitats for all kinds of bacteria, including pathogens, and maybe responsible for contaminations of bulk water systems.Nowadays, DNA-based methods are used for the detection andcharacterization of bacteria. One of the major disadvantages of thesetechniques is that they can not distinguish between DNA from live and deadcells. A battery of methods to face this problematic is presented in this work.Conditioned surface water disinfected with ozone/ClO 2 flowed through apilot scale built up with different pipe materials for biofilm formation.Bacterial population analysis was done by PCR-DGGE, comparing directsamples (total DNA) and samples pre-treated with Propidium monoazide orDNase I (DNA from live cells). Shifts in the DNA patterns observed afterDGGE analysis, demonstrated: (i) the applicability of PMA and DNase Itreatment in natural biofilm investigation; (ii) detection of DNA from deadbacteria and eDNA was blocked by pretreatment with PMA or DNase I; and(iii) DNase I treatment demonstrated a clearer effect on live/deaddifferentiation. Traditional cultivation methods and qPCR completed thebiofilm analysis.The results of the bacterial population analysis and the results of thequantification methods that provide an overview of the differentphysiological states of bacteria: live cells, total amount of cells, andcultivable cells, are presented here.spektrum | Tagungsband 2011