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

224SMP044RNase J and RNase E in Sinorhizobium meliloti: specific andcommon roles in rRNA maturation, RNA modification,motility and quorum sensingK. Baumgardt 1 , S. Thalmann 1 , R. Madhugiri 1 , A. Schikora 2 , K.-H. Kogel 2 ,G. Klug 1 , A. Becker 3 , E. Evguenieva-Hackenberg* 11 Justus-Liebig-Universität Giessen, Institut für Mikrobiologie undMolekularbiologie, Gießen, Germany2 Justus-Liebig-Universität Giessen, Institute of Phytopathology andApplied Zoology, Gießen, Germany3 Albert-Ludwigs-Universität Freiburg, Institute of Biology III, Freiburg,GermanySinorhizobium meliloti Rm2011, a nitrogen-fixing plant symbiont,harbours RNase E and RNase J, two principal RNases of Bacillus subtilisand Escherichia coli, respectively (1). To address the mechanisms forposttranscriptional regulation of gene expression inS. meliloti, weanalyzed mutants with mini-Tn5 insertions in the genes encoding RNase Eand RNase J (2) in comparison to the wild type. Only the RNase J mutantbut not the RNase E mutant was impaired in growth, motility and rRNAmaturation (3). However, small RNAs, tRNAs and mRNAs were affectedin both mutants. Small RNAs and tRNAs have identical lengths butmigrate differently in denaturing gels when compared to the wild type,suggesting hypermodification of RNA in the two mutants. Consistent withthis, the gene SMc00649 encoding a probable RNA methylase was upregulatedin the mutant strains. Additional microarray and qRT-PCRanalyses revealed specific and overlapping effects on mRNA level. Thedetected down-regulation of genes involved in motility and chemotaxis inthe two mutants suggested differences in the quorum-sensing response incomparison to the wild type. Indeed, production of AHLs was increased inthe mutant strains, while overproduction of RNase E resulted in a strongdecrease of the AHL amounts. Analysis of genes involved in AHLs productionshowed that balanced expression of RNase E ans RNase J is important for theposttranscriptional control of quorum sensing in S. meliloti.1. Evguenieva-Hackenberg E, Klug G. (2009) New aspects of RNA processing in prokaryotes.Curr. Opin.Microbiol. 14(5):587-92.2. Pobigaylo, N, Wetter, D, Szymczak, S, Schiller, U, Kurtz, S, Meyer, F, Nattkemper, TW, Becker, A.(2006) Construction of a large signature-tagged mini-Tn5 transposon library and its application tomutagenesis ofSinorhizobium meliloti.Appl Environ Microbiol, 72, 4329-4337.3. Madhugiri R, Evguenieva-Hackenberg E. (2009) RNase J is involved in the 5'-end maturation of 16SrRNA and 23S rRNA inSinorhizobium meliloti.FEBS Lett, 583, 2339-2342.SMP045Change of microbial community composition due togeothermal use of the subsurfaceA. Westphal* 1 , A. Jesußek 2 , M. Alawi 1 , A. Dahmke 2 , H. Würdemann 11 GeoForschungsZentrum Potsdam, ICGR, Potsdam, Germany2 Universität Kiel, Angewandte Geowissenschaften, Kiel, GermanySeasonal heat storage systems for district heating and buildingclimatisation are of increasing importance to secure a sustainable energyuse and supply. For an efficient and permanent reliable use of geothermalenergy the impact on the environment has to be evaluated.Our presentation encompasses a study of a lab-scale column experiment toquantify the effects of different temperatures on solution, precipitation andmicrobially catalysed redox processes.Four different tempered columns (10, 25, 40, 70°C) were operated andsodium actetate was added continuously. To characterize the microbialbiocenosis of the initial sediment samples, fluid samples from the upperexit and also over the profile (9 sampling ports) were collected. Allsamples were analysed based on partial 16S rDNA. Among fingerprintingmethods (PCR-DGGE) for the characterization of the microbialbiocenosis, qPCR and FISH will be applied for the quantification ofmicroorganisms and the determination of their metabolic activity.Sulfate reduction in all columns was detected with the highest reductionrates at 40°C. First fingerprinting results show a shift of the dominantmicroorganisms due to the different temperatures. Additionally, themicrobial composition in the 10°C column changed clearly in between thedifferent sampling ports of the column. Methane production was measured at25°C correlating with Archaea occurrence.Lab-scale column experiments showed an alteration in the microbial biocenosisdue to geothermal induced temperature effects. The identification ofmicroorganisms enables the correlation to metabolic classes and providesinformation about biochemical processes in the used groundwater system andtherewith the impact on plant operation as well as environment.Consequently, plasmids play a major role in enhancing the geneticdiversity and adaptation of bacteria as agents of horizontal gene transfer(HGT). IncP-9 plasmids are very important vehicles for degradation andresistance genes that are assumed to contribute to the adaption of bacterialcommunities in environments contaminated with xenobiotic compoundssuch as biofilters that are used for microbial degradation of pesticides. Inthis study the abundance and diversity of IncP-9 plasmids in six differentbiofilters (three replicates per biofilter) from Belgium was analyzed.Anewly developed primer system targeting therep-oriVregion (S1: 610 bp)was used for PCR amplification from total community DNA. Southern blothybridization confirmed the presence of IncP-9 plasmid specific sequencesin all biofilters with one exception. In order to obtain insights into thediversity of the IncP-9 plasmids, amplicons obtained from biofilters 1(Leefdaal), 2 (Pcfruit) and 5 (Kortrijk), that showed the strongest IncP-9signals, were cloned and sequenced. In addition, a quantitative real timePCR system (S2: ~200 bp) was established in order to quantify IncP-9abundance. The log10 transformed ratio of IncP-9 to16S rRNA genecopies varied from -3.1 to -2.65. To validate the specificity of the qPCRprimer system, the amplicons were also cloned and sequenced. Bothsystems specifically amplified IncP-9 sequences from total communityDNA. While all sequences amplified with both primer systems frombiofilter 5 showed a high sequence similarity to IncP-9 (pNL15),sequences obtained from biofilter 1 and biofilter 2 were more diverse,affiliated to the IncP-9a (pMT3 - biofilter 2), IncP-9b (pWW0 - biofilter1), IncP-9d (pNAH7; only obtained with S1- biofilter 2) and IncP-9 (pNL15 - biofilters 1 and 2). In addition several novel sequences onlydistantly affiliated to the clusters defined by Sevastsyanovich et al., 2008were obtained from biofilters 1 and 2. Interestingly only a small number ofidentical sequences were picked up with both systems indicating asurprisingly high sequence diversity of IncP-9 plasmids in biofilters.This work was supported by the EU project METAEXPLORE and the DFG project SM59/8-1Sevastsyanovich, Y.R., R. Krasowiak, L.E.H. Bingle, A.S. Haines, S.L. Sokolov, I.A. Kosheleva,A.A. Leuchuk, M.A. Titok, K. Smalla, and C.M. Thomas.2008. Diversity of IncP-9 plasmids ofPseudomonas. Microbiology 154, 2929-2941.SMP047Minerals and charcoal - factors shaping microbial communitycomposition and bacterial response to phenanthrene inartificial soilsD. Babin* 1 , G.-C. Ding 1 , G.J. Pronk 2 , H. Heuer 1 , K. Heister 2 , I. Kögel-Knabner 2 , K. Smalla 11 Julius Kühn-Institut, Bundesforschungsinstitut für Kulturpflanzen, Institut fürEpidemiologie und Pathogendiagnostik, Braunschweig, Germany2 Technische Universität München, Lehrstuhl für Bodenkunde, Freising,GermanyIn soil, different organic, inorganic and biological constituents arecontacting each other and forming large biogeochemical interfaces. Theirinteractions are poorly understood and therefore this study explored theinfluences of soil minerals and charcoal on microbial communities. Due toproblematic comparison of natural soils, in a microcosm experiment sevenartificial soils were composed varying in clay minerals (illite,montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal. Thesame aliquots of the microbial fraction extracted from Cambisol were usedas initial microbial community and autoclaved manure as nutrient sourcefor each artificial soil. Incubation took place under constant environmentalconditions up to 18 months (sampling on day 1, 9, 31, 90, 180, 460, 450).Total community DNA was extracted and the 16S rRNA gene and ITSamplicons for Bacteria or Fungi, respectively, were used in denaturinggradient gel electrophoresis (DGGE) to generate molecular fingerprints.DGGE analysis showed that mineral composition and charcoal influencethe establishment of microbial communities in artificial soils, even after along incubation time. Especially the charcoal soil showed pronounceddifferences in the DGGE pattern compared to other artificial soils withoutcharcoal.To explore the response of the established microbial communities topersistent organic pollutants, one-year old artificial soils were spiked withphenanthrene (2 g/kg) and incubated for another 70 days. By DGGE, shiftsin the bacterial but not fungal communities were revealed between nonspikedand phenanthrene-contaminated samples. Interestingly, bacterialcommunities of different artificial soils showed distinct phenanthreneresponses. By plating, higher bacterial counts were found in soils treated withphenanthrene.In conclusion, minerals and charcoal in artificial soils shaped the compositionof microbial communities and the bacterial response to phenanthrene.SMP046Characterization of IncP-9 in different biofilters from Belgiumcontaminated with pesticidesS. DealtryJulius Kühn-Institut , EP, Braunschweig, GermanyConjugative plasmids seem to be one of the mobile genetic elements mostresponsible for the rapid adaptation to environmental selective pressure.BIOspektrum | Tagungsband 2012