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

MDP016Intrinsic differences in denitrifier community structureand abundance determine functional responses ofdenitrification in three organic soilsK. Brenzinger* 1 , G. Braker 1 , P. Dörsch 2 , L. Bakken 21 Department of Biogeochemistry, Max Planck Institute for TerrestrialMicrobiology, Marburg, Germany2 Norwegian University of Life Sciences, Aas, NorwayDenitrification is an alternative anaerobic respiration process reducingnitrogen oxides (NO 3 - and NO 2 - ) stepwise to N 2 via the intermediates NOand N 2O. This process completes the global nitrogen cycle and is ofparticular importance for the biogeochemical cycling of nitrogen in soils.Soils are important sources for N 2O, a potent greenhouse gas and contributeabout 70% of the N 2O emitted to the atmosphere. The microorganismscapable of denitrification are polyphyletic and exhibit differences in theinduction and activity of the denitrification system in individual strainswhich could result in ecosystem level differences in N 2O emission underdifferent conditions [4]. Thus, community composition will affectcommunity and ecosystem functioning.In this study, we comparatively evaluated the structure and abundance aswell as the similarity of denitrifier communities from three drained organicsoils in Finland, Germany and Sweden differing in soil history and soilparameters. Structure and abundance of denitrifier communities wereexplored based on their NO 2 - -reductase (nirK/nirS) and N 2O-reductase(nosZ) genes as proxies for the ability of the communities to produce andreduce N 2O. We hypothesized that the denitrifier communities harbored bythese soils were composed differently since marked physiologicaldifferences in denitrification response to anoxia [2] and low temperature [1,3] occurred. Moreover, a direct effect of pH had been observed whenexposing bacterial consortia extracted from these soils to two different pHlevels (pH 5.4 and 7.1). We evaluated differences in the diversity,composition and abundance of denitrification genotypes between soils andconclude that links exist between the genetic makeup and physiologicalresponses across the three denitrifier communities. Moreover, wehypothesize that functional differences were enhanced due to differences inthe composition of the active denitrifier community in response to differentenvironmental triggers, e.g. temperature and pH.[1] Dörsch, P. and L.R. Bakken (2004): Low-temperature response of denitrification: Comparison ofsoils. Eurasian Soil Science 37: S102-S106.[2] Holtan-Hartwig, L. et al (2000): Comparison of denitrifying communities in organic soils: kineticsof NO3 - and N2O reduction. Soil Biol Biochem 32: 833-843.[3] Holtan-Hartwig, L. et al (2002): Low temperature control of soil denitrifying communities:kinetics of N2O production and reduction. Soil Biol Biochem 34: 1797-1806.[4] Schimel, J.P. and J. Gulledge (1998): Microbial community structure and global trace gases.Global Change Biol 4: 745-758.MDP017A novel approach for alphaprotebacterial plasmidclassificationO. Frank*, N. Buddruhs, V. Michael, O. Päuker, S. Pradella, J. PetersenMolecular Systematics, German Collection of Microorganisms and CellCultures (DSMZ), Braunschweig, GermanyMembers of the Roseobacter clade are endowed with a remarkable wealth ofplasmids, e.g. up to twelve extrachromosomal replicons could be identifiedin Marinovum algicola (Pradella et al. 2009), comprising one third of thetotal genomic information. To investigate this diversity, a comprehensiveplasmid classification scheme was established.Plasmids are classified according to their compatibility, i.e. the ability oftwo or more plasmids to be stably maintained in a cell lineage. Ourclassification approach is based on phylogenetic analyses of the replicationoperons, which constitute the functional backbone of plasmids. Thesesystems comprise the genes for replication and partitioning, revealing acommon evolutionary history due to functional linkage. Operons of the sametype can be found on up to four plasmids in a single cell, indicating theircompatibility. The required functional divergence of compatible plasmidscorrelates with phylogenetic distance, i.e. their replication modules arelocated in different subtrees. In case of the alphaproteobacterial replicationoperon repABC, nine distinct groups were identified in the Roseobacterclade (Petersen et al. 2009). To validate our phylogeny based in silicopredictions regarding plasmid compatibility, we developed a test system:selected repABC - modules were cloned into suited vectors and introducedinto Phaeobacter gallaeciensis DSM 17395 . Successful transformation canbe traced through specific antibiotic resistances provided by the respectiveconstruct. Accordingly, double transformands can be detected by theexpressed double resistance resulting from maintenance of compatibleconstructs.We observed that plasmids with phylogenetically closely related repABCoperons, outcompete each other, and are therefore incompatible. In contrast,plasmids with distant repABC operons, stably coexist in the cell and arecompatible. The results verify the predictions deduced from the in silicoanalyses.Hence, our phylogenetic classification framework for plasmid replicationsystems allows the rapid allocation of new plasmids from incoming genesequences. Furthermore it allows the development of genetic tools for entireplasmid knockouts and the comparison of plasmid knockout mutants andwild type strains will reveal the significance of alphaproteobacterialplasmids.Petersen J, Brinkmann H, Pradella S (2009) Diversity and evolution ofrepABC-type plasmids in Rhodobacter. Environ MicrobiolPradella S, Päuker O, Petersen J (2009) Genome organisation of the marineroseobacter clade member Marinovum algicolaMDP018Flavobacteria of the North Sea: Diversity of CulturabilityR. Hahnke*, J. HarderDepartment of Microbiology, Max Planck Institute for MarineMicrobiology, Bremen, GermanyFlavobacteria account according to cultivation-independent in situhybridisation experiments for up to 30% of the Bacteria in the North Sea.They are considered as ecologically important microorganisms involved inthe degradation of polymers. So far, only a few isolates have been describedfrom the North Sea, mainly Maribacter (Barbeyon et al., 2008) andDokdonia (Riedel et al., 2010). But 16S rRNA gene clone libraries havesuggested that other species are the ecologically significant Flavobacteria.We attempted a cultivation of Flavobacteria from different North Seahabitats (Harlesiel, Helgoland, Janssand, Sylt) originating from differentmarine sample materials (sediment, seawater and surfaces of plants, animalsand stones) on agar plates with a variety of carbon sources (malate, glucose,arabinaose, cellobiose, galactose, xylose, peptone, casaminoacids, yeastextract) and sometimes the antibiotic kanamycin. Candidate colonies wereidentified by their yellow to orange colour and rod-shaped morphologyunder the microscope. Subsequently, 483 isolates were screened by PCRwith a Flavobacteria specific primer designed for this purpose and thepartial 16S rRNA gene was sequenced, revealing 307 Flavobacteria, 2Sphingobacteria and 11 Cytophagia. The strains affiliated with 24 genera.Furthermore, representative isolates were analysed for flexirubin typepigments. Comparable to the literature, isolates affiliated with the generaZobellia, Grigella, and Aquamarina were flexirubin positive. But someisolates of the genera Arenibacter and Lacinutrix were flexirubin negative,in contrast to the literature. In this study we were able to isolate strains ofnovel species of the Flavobacteria originating from the North Sea. A firstanalysis revealed a distinction between pelagic and costal isolates, as well asbetween isolates from sediment and sea water.MDP019Comparative phenomics of the wild type Phaeobactergallaeciensis and its 65 kb plasmid knock-out mutantN. Buddruhs*, O. Frank, V. Michael, O. Päuker, S. Pradella, J. PetersenMolecular Systematics, German Collection of Microorganisms and CellCultures (DSMZ), Braunschweig, GermanyPlasmids of the marine Roseobacter clade carry important genetic traits, likegenes for the aerobic anoxygenic photosynthesis or the catabolism ofphenylacetate [3, 4]. Our completely sequenced model organismPhaeobacter gallaeciensis DSM 17395 harbours three plasmids with sizesof 262 kb, 78 kb and 65 kb. The smallest plasmid includes a conspicuouswealth of genes for polysaccharide metabolisms, e.g. for mannose andrhamnose synthesis. The same polysaccharides are involved in symbioticadhesion of Rhizobia [1] and may also be responsible for biofilm formationof P. gallaeciensis and symbiotic interactions with algae.To investigate the function of the 65 kb plasmid, we generated the respectiveknock-out mutant based on plasmid incompatibility. Extrachromosomalelements harbour specific modules for autonomous replication andpartitioning systems, but similar systems are incompatible and theseplasmids cannot coexist within the same cell. The 65 kb plasmid contains aRepA-replication system and we cloned the homologous module fromspektrum | Tagungsband 2011