Symbiotic Interactions Poster - Helmholtz Zentrum München · Insect ‐ Nematode ‐ Invertebrate...

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Poster Abstracts

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Poster group 1 (PG1): Humans ‐ Animals

PG1/1 Combined approach to improve the genetic accessibility of Bifidobacterium bifidum V.F. Brancaccio, E. Kashani, D. Zhurina, C.U. Riedel PG1/2 Adhesion of B. bifidum to intestinal epithelial cells is mediated by proteinaceous cell wall components Marita Gleinser and Christian U. Riedel PG1/3 Treatment with a B. bifidum strain ameliorates colitis in the in Rag1‐/‐ transfer model Julia Preising, Jan‐Hendrik Niess, Christian U. Riedel PG1/4 HYDRA – A COMMUNITY AFFAIR S. Fraune and T.C.G. Bosch PG1/5 Dynamic regulation of N‐acyl‐homoserine lactone production and degradation in Pseudomonas putida IsoF Michael Rothballer, Agnes Fekete, Christina Kuttler, Doreen Fischer, Katharina Buddrus‐Schiemann, Burkhard A. Hense, Marianna Lucio, Johannes Müller, Philippe Schmitt‐Kopplin and Anton Hartmann PG1/6 Magnetic Nanoparticles as Extraction Method to Analyse Homoserine Lactones by Ultra‐Performance Liquid Chromatography (UPLC) Juliano R. Fonseca, Agnes Fekete, Philippe Schmitt‐Kopplin

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Poster group 2 (PG2):

Insect ‐ Nematode ‐ Invertebrate PG2/1 Transmission route and population dynamics of a defensive insect symbiont Martin Kaltenpoth, Wolfgang Goettler, Sabrina Köhler, Erhard Strohm PG2/2 Exploring prokaryotic‐microfauna interactions: a case study on the polyextremophile microfauan species Tardigrada N. M. Lee, I. Jansson, O. Braissant, U. Baumann, Beiying Dai, P. Rettberg, G. Horneck PG2/3 INVESTIGATING THE DIVERSITY AND ECOLOGY OF EPIBIOTIC BACTERIA ON FRASASSI CAVE‐DWELLING AMPHIPODS: MOLECULAR EVIDENCE FOR HOST‐SYMBIONT SPECIFICITY Jan Bauermeister, Jean‐François Flot, Sharmishtha Dattagupta PG2/4 WHOLE GENOME AMPLIFICATION OF A SINGLE PORIBACTERIAL CELL: INSIGHTS INTO THE GENOME OF A MARINE SPONGE SYMBIONT Alexander Siegl and Ute Hentschel

Poster group 3 (PG3): Plant ‐ Soil

PG3/1 Root colonization by Pseudomonas sp. DSMZ 13134 and effect as biological plant growth stimulant in barley Katharina Buddrus‐Schiemann, Michael Schmid, Anton Hartmann PG3/2 UPTAKE AND TRANSPORT OF N‐ACETYL‐HOMOSERINE LACTONES IN BARLEY Tina Riedel, Sandor Forczek, Miroslav Matucha, Peter Schröder PG3/3 Evidence for a plant associated natural habitat of Cronobacter spp. Michael Schmid, C. Iversen, I. Gontia, R. Stephan, A. Hofmann, A. Hartmann, B. Jha, L. Eberl, K. Riedel, A. Lehner PG3/4 HALOTOLERANT PGPR FROM SALICORNIA BRACHIATA, AN EXTREME HALOPHYTE Iti Gontia, Bhavanath Jha, Michael Schmid, and Anton Hartmann

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PG3/5 TRANSCRIPTIONAL RESPONSES OF BRADYRHIZOBIUM JAPONICUM TO ENVIRONMENTAL CHANGES Kathrin Lang, Mandy Wenzel, Gabriella Pessi, and Michael Göttfert PG3/6 SALIX CAPREA RHIZOBACTERIA AND ENDOPHYTES WITH POTENTIAL TO ENHANCE EFFECTIVENESS OF HEAVY METAL PHYTOEXTRACTION FROM SOIL K. Fallmann, M. Kuffner, M. Puschenreiter, G. Wieshammer, S. dos Reis and A. Sessitsch PG3/7 3‐Methylarginine, a toxin from the symbiotic epiphyte Pseudomonas syringae pv. syringae 22d/93 Janine Hofmann, Sascha D. Braun, Beate Völksch, and Dieter Spiteller PG3/8 STRUCTURE AND FUNCTION OF POTATO ASSOCIATED MICRORGANISMS IN THE CENTRAL ANDEAN HIGHLANDS Stefan Pfeiffer, Branislav Nikolic and Angela Sessitsch PG3/9 Microbial Endophytes From Warburgia ugandensis ‐ Diversity and Effects on Host Drimane Sesquiterpenes Birgit Mitter, Sigrid Drage, Christina Tröls, Franz Hadacek, Alice Muchugi, Ramni Jamnadass and Angela Sessitsch

PG3/10 EFFECTS OF TRANSGENIC MAIZE ON BENEFICIAL PLANT‐MICROBE‐INTERACTIONS AND SOIL NITROGEN CYCLING Michaela Prischl, Evelyn Hackl and Angela Sessitsch PG3/11 Risks and Recommendations Regarding Human Pathogens in Organic Vegetable Production Chains (PATHORGANIC) Fenzl C, Hackl E, Brankatschk K, Jäderlund L, Jensen A., Hofmann A., Rinnofner T., Koller M., Friedel J., Duffy B, Arthurson V., Wyss G., Schmid M., Baggesen D. and Sessitsch, A. PG3/12 METAGENOMIC ANALYSIS OF 1‐AMINOCYCLOPROPANE‐1‐CARBOXYlATE (ACC) DEAMINASE GENES AMONG POTATO ENDOPHYTES Branislav Nikolic, Helmut Schwab, Angela Sessitsch PG3/13 HYDROCARBON DEGRADATION AND PLANT COLONIZATION OF SELECTED BACTERIAL STRAINS ISOLATED FROM ITALIAN RYEGRASS AND BIRDSFOOT TREFOIL Sohail Yousaf, Katrin Ripka, Verania Andria, Thomas Reichenauer, Muhammad Afzal and Angela Sessitsch

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Plant ‐ Soil; Water ‐ Waste PG4/1 DNA BARCODING AND SEQUENCE BASED IN‐FIELD SPECIES DETECTION OF ARBUSCULAR MYCORRHIZAL FUNGI Manuela Krüger, Herbert Stockinger, Arthur Schüßler PG4/2 MOLECULAR SIGNALLING IN THE TRIPARTITE SYMBIOSIS OF PIRIFORMOSPORA INDICA – RHIZOBIUM RADIOBACTER‐BARLEY Li, D., Sharma, M., Zuccaro, A., Fekete, A., Schmid, M., Rothballer, M., Schmitt‐Kopplin, P., Kogel, K.‐H., and Anton Hartmann PG4/3 Symbiotic based cultivation for improved cultivation strategy of novel species within the phylum Chloroflexi N. M. Lee; D. B. Meisinger, K. M. Ritalahti, S. Spring, M. Schmid, F. E. Löffler PG4/4 BIOLOGICAL ROLE OF SECONDARY METABOLITES FROM MARINE MICROORGANISMS Heike I. Baumann, Jutta Wiese, Andrea Gärtner, Franz Goecke, Herwig Heindl, Inga Kajahn, Katrin Kleinschmidt, Antje Labes, Kerstin Nagel, Sven Neulinger, Birgit Ohlendorf, Rolf Schmaljohann, Imke Schneemann, Dirk Schulz, Tim Staufenberger, Rüdiger Stöhr, Vera Thiel, Heidi Zinecker and Johannes F. Imhoff PG4/5 MOLECULAR AND PHYSIOLOGICAL CHARACTERIZATION OF THE CLOSE BACTERIAL SYMBIOSIS IN PHOTOTOPHIC CONSORTIA Johannes Müller, Roland Wenter, Dörte Dibbern, Veronika Reisinger, Matthias Plöscher, Lutz Eichacker and Jörg Overmann PG4/6 ECOPHYSIOLOGY AND CELL‐CELL‐INTERACTION OF A NOVEL MULTICELLULAR MAGNETOTACTIC PROKARYOTE FROM NORTH SEA SEDIMENTS Roland Wenter, Gerhard Wanner, Dirk Schüler and Jörg Overmann PG4/7 A ROLE FOR OLIGOCHAETE‐ENDOSYMBIOTIC MICROBES IN SULPHUR‐CYCLING IN CONTAMINATED AQUIFERS? Giovanni Pilloni, Tanja Riedel, Kathrin Euringer, Claudia Kellermann and Tillmann Lueders PG4/8 Homogeneous inoculation vs. microbial hot spots of isolated strain and microbial community: What is the most promising approach in remediating soils contaminated with organic chemicals? A. Krug, F. Wang, U. Dörfler, D. Fischer, M. Schmid, J.C. Munch, R. Schroll

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Poster Abstracts:

Poster group 1 (PG1): Humans ‐ Animals ‐ Archaea

PG1/1

Combined approach to improve the genetic accessibility of Bifidobacterium bifidum

V.F. Brancaccio, E. Kashani, D. Zhurina, C.U. Riedel

Institute of Microbiology and Biotechnology, University of Ulm We recently demonstrated the good anti‐inflammatory capacity of a potential probiotic strain of B. bifidum in the Rag1‐/‐ transfer model of murine colitis. However, studies of this and other probiotic candidate strains of bifidobacteria at the molecular level are limited by the lack of molecular tools and the low transformation efficiency. To improve the genetic accessibility of B. bifidum, the transformation protocol was optimized for buffer conditions and electrical pulse settings by transforming the E. coli‐Bifidobacterium shuttle vector pMDY23. To further improve the transformation efficiency, the restriction barrier for foreign DNA by restriction‐modification (R‐M) systems should be overcome. Using in silico analysis one type I and one type II RM system were predicted in the genome of our B. bifidum strain. Their presence was confirmed by the PCR and Southern Blotting and both systems were shown to be expressed by RT‐PCR. The type II methyltransferase was subcloned into pIMK4 vector for IPTG‐inducible overexpression in E. coli Top10. This should allow efficient methylation of any shuttle vectors in this strain prior to the transformation into B. bifidum thereby circumventing the restriction barrier to foreign DNA in this species.

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PG1/2 Adhesion of B. bifidum to intestinal epithelial cells is mediated by proteinaceous cell wall

components

Marita Gleinser and Christian U. Riedel

Institute of Microbiology and Biotechnology, University of Ulm We analyzed the adhesive structures of a potential probiotic B. bifidum strain that showed excellent adhesion to IECs in previous studies as well as potent anti‐inflammatory activity. Clean cell wall, membrane and cytoplasmic fraction were prepared. In particular, the cell wall fraction inhibited adhesion of whole cells of a B. bifidum strain to differentiated IECs. To identify the chemical nature of the structures responsible for adhesion, whole cells of our B. bifidum strain were treated with pronase, lipase and periodate. After pronase treatment several bands were absent in the cell wall fraction from bacteria compared to the cell wall fraction from untreated B. bifidum. Furthermore, treatment of a B. bifidum strain with pronase significantly decreased adhesion to IECs whereas treatment with lipase and periodate did not have an effect on adhesion. This indicates that proteinaceous cell surface components are involved in adhesion of B. bifidum to IECs. Recently, the cell surface protein BopA was shown to be involved in adhesion of B. bifidum to IECs. BopA was expressed in E. coli BL21 and adhesion assays were performed with purified protein. Additionally, B. bifidum strains with altered expression of BopA will be generated to further analyse the impact of BopA on adhesion.

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PG1/3

Treatment with a B. bifidum strain ameliorates colitis in the in Rag1‐/‐ transfer model

Julia Preising, Jan‐Hendrik Niess, Christian U. Riedel

Institute of Microbiology and Biotechnology, University of Ulm

Here we compare the anti‐inflammatory effects of a strain of B. bifidum with excellent adhesive properties and potent anti‐inflammatory capacity in vitro in the Rag1‐/‐ CD4+ T‐cell transfer model of murine colitis to a non‐adherent strain with no anti‐inflammatory capacity. In a placebo‐controlled set up two groups of Rag1‐/‐ mice (n = 3 per group) received one oral dose of either the probiotic B. bifidum or the ‘non‐probiotic’ B. longum/infantis strain (each at 2×109 cfu per animal) followed by transfer of CD4+ T‐cells. Two control groups received placebo of which one group also was transferred with CD4+ T‐cells to induce colitis. Feeding with the probiotic and placebo was continued three times a week until the end of the trial when all animals were sacrificed. The anti‐inflammatory effect of feeding the B. bifidum strain was assessed by measuring weight and length of dissected colons, histology scores of colonic tissue samples, and qRT‐PCR for a pro‐inflammatory cytokine (interleukin‐6). Treatment with the probiotic significantly improved colitis as measured by these parameters whereas B. longum/infantis had no effect.

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PG1/4

HYDRA – A COMMUNITY AFFAIR

S. Fraune & T.C.G. Bosch

Zoological Institute, Christian‐Albrechts‐University Kiel, Germany

Epithelia in all animals, which are exposed to the environment, are colonized by more or less diverse communities of microbes. Since Hydra is an early‐branching metazoan and has preserved much of the genetic complexity of the common metazoan ancestor it promises to be highly informative to discover evolutionary conserved mechanisms controlling epithelial host‐microbe interactions. We previously have shown that the hydra epithelium actively selects and shapes its microbial community indicating distinct selective pressures imposed on and within the epithelium. Here, we present two recent observations which may have profound impact on understanding the maintenance of species‐specific bacterial microbiota with its hosts. (i) Hydra in which tissue homeostasis was experimentally disturbed by eliminating distinct cell types from the epithelium display significant differences in the microbial communities before and after loss of cell homeostasis. (ii) Intriguingly, overexpression of antimicrobial peptides in ectodermal epithelial cells of polyps has drastic effects on the bacterial community resulting in reduced bacterial load and changes in the composition of the colonizing microbiota. Together, these insights indicate a link between host tissue and colonizing microbiota, and promise to unveil ancient mechanisms derived from the in vivo context of a whole epithelial organism that control tissue and host‐microbe homeostasis.

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PG1/5

Dynamic regulation of N‐acyl‐homoserine lactone production and degradation in Pseudomonas putida IsoF

Michael Rothballer3*, Agnes Fekete1*, Christina Kuttler4*, Doreen Fischer3, Katharina

Buddrus‐Schiemann3, Burkhard A. Hense2, Marianna Lucio1, Johannes Müller4, Philippe Schmitt‐Kopplin1 and Anton Hartmann3

Helmholtz Zentrum München, German Research Centre for Environmental Health (GmbH), 1Institute of Ecological Chemistry, 2Institute of Biomathematics and Biometry, 3Department

of Microbe‐Plant Interactions, Ingolstädter Landstr. 1, D‐85764 Neuherberg/Munich, Germany, 4Technical University Munich, Centre for Mathematical Sciences, Boltzmannstr. 3,

85748 Garching/Munich, Germany *These authors contributed equally to this work

Bacteria in microcolonies as well as in biofilms are regularly attached to and interact with other organisms, such as plant roots or fungi. The N‐acyl‐homoserine lactone (AHL) production of these bacterial assemblages has been shown to influence the behaviour of the colonized organisms considerably, which is termed cross‐kingdom signalling. For a better understanding of this interaction and the impact on the host it is necessary to learn more about the time dependent quantitative changes of the AHL signalling molecules. Therefore, the biocontrol strain Pseudomonas putida IsoF, which was isolated from a tomato rhizosphere and is a known AHL‐producer with only one LuxI/LuxR like quorum sensing system, was analysed for its AHL production in different growth phases. With the analytical tools of ultra performance liquid chromatography and high resolution mass spectrometry it was possible to determine not only the various AHLs synthesized over time but also their degradation products. 3‐oxo‐decanoyl‐homoserine lactone was found to be the dominant AHL which reached its maximum already in the early logarithmic growth phase. Although the pH of the medium was neutral, the AHLs were degraded thereafter rapidly to the corresponding homoserines and other metabolites. The proposed lactonase gene of P. putida IsoF could not be identified, because it is apparently quite different to hitherto described lactonases. The analytical data were used to calculate rates and thresholds of AHL production by mathematical modelling allowing quantitative predictions and further understanding of the quorum sensing based regulations in this bacterium. This study combining microbiological, chemical and mathematical approaches suggests that AHL degradation is an integral part of the whole autoinducer circuit of P. putida IsoF.

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PG1/6 Magnetic Nanoparticles as Extraction Method to Analyse Homoserine Lactones by Ultra‐

Performance Liquid Chromatography (UPLC)

Juliano R. Fonseca, Agnes Fekete, Philippe Schmitt‐Kopplin

Helmholtz Zentrum München, Department of BioGeoChemistry and Analytics, IÖC

Derivatives of N‐acylhomoserine lactones (HSLs) occur as quorum or diffusion sensing molecules in Gram‐negative bacteria and their quantitative chemical analysis became important as a possible way to follow regulation processes of their pathogenicity towards plants, animals and in health.

N‐acyl‐homoserine lactones consist of a furen‐2‐on group with an acyl chain contain of 4 to 14 (less frequently up to 18) carbons. Unsubstituted HSLs have a LogP between 0.03 for C4‐HSL to 5.09 for C14‐HSL. HSLs can be substituted at the β‐carbon by a keto‐ or hydroxyl‐function and the fatty acid side chain may be unsaturated.

Recently, magnetic materials have been developed due their potential applications in cell separation, protein isolation and enzyme immobilization. With this in mind, nanoparticles combining magnetic iron oxide and octadecyl‐silica microspheres was prepared and evaluated as extraction technique. The results show that this material can be used for the isolation of homeserine lactones in solution. As expected, significant differences in respect to adsorption capacity were observed regarding different HSLs chain size. Using an UPLC‐UV system, it has been possible to separate 15 HSLs in a short time by reverse phase chromatography (total run time 3.5 min/sample) with RSD of area less than 10% and for retention time, less than 1%. Interestingly, in the case of oxo‐homoserine lactones, the occurrence of keto‐enol tautomerism was observed and studied as well. The peak shape was influenced by pH where α peak changes from 3 at pH 2 to 1.4 at pH 10.

Literature: Fekete, A. et al, Bioanal Chem. 2007, 387, 455–467.

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Insect ‐ Nematode ‐ Invertebrate

PG2/1

Transmission route and population dynamics of a defensive insect symbiont

Martin Kaltenpoth1,2,3, Wolfgang Goettler2, Sabrina Köhler2, Erhard Strohm2

1Max Planck Institute for Chemical Ecology, Hans‐Knöll‐Str. 8, 07745 Jena, Germany 2University of Regensburg, Department of Zoology, 93040 Regensburg, Germany

3University of Utah, Department of Human Genetics, 20 South 2030 East, Salt Lake City, UT 84112, USA

Insects engage in mutualistic relationships with a wide variety of microorganisms that are usually transmitted vertically to the next generation. During transmission, the symbiont populations often suffer significant bottlenecks that may entail major genetic and genomic consequences. Here we investigated the transmission route and population dynamics of a symbiont with an unusual way of post‐hatch vertical transmission by using quantitative PCRs and morphological 3D‐reconstructions. European beewolves (Philanthus triangulum, Hymenoptera: Crabronidae) harbor symbiotic bacteria (‘Candidatus Streptomyces philanthi’) in specialized antennal gland reservoirs and secrete them into their subterranean brood cells. The symbionts are later taken up by the beewolf larva and incorporated into the cocoon material to provide protection against pathogenic microorganisms. Even after months of hibernation, the symbiont population on the cocoon is estimated to encompass around 1.4x105 cells. However, our results indicate that only few of these bacterial cells (about 9.7x102) are taken up from the cocoon by the emerging female. The symbiont population subsequently undergoes logistic growth within the antennal gland reservoirs and reaches a maximum of about 1.5x107 cells three to four days after emergence. The maximum specific growth rate is estimated to be 0.084 to 0.105 h‐1. With a total reduction in cell numbers of about 6.7x10‐5 during vertical transmission, the symbiont population experiences one of the most severe bottlenecks known for any symbiotic system to date, which may have significantly affected the evolution of the beewolf‐Streptomyces symbiosis.

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PG2/2

Exploring prokaryotic‐microfauna interactions: a case study on the polyextremophile microfauan species Tardigrada

N. M. Lee[1], I. Jansson[2], O. Braissant [3], U. Baumann[1], Beiying Dai[1], P. Rettberg[4],

G. Horneck[4]

[1]Microbiology, Techn. Univ. Mnchen, Freising, Germany [2]Microbiology, Genetics, Toxicology, Stockholm University, Sweden

[3]Lab. for Biomechanic and Biocalorimetry, University of Basel, Basel, Switzerland [4]DLR, German Aerospace Center, Institute of Space Medicine, Division of Radiation

biology/Astrobiology, Cologne, Germany R. Amann stated in his well‐cited review from 1995 that "consideration of symbionts co‐evolving with arthropods like insects may expand the number of extant bacterial species by several orders of magnitude" (Microbiol. Rev, 59, 1, 143). Since this statement, several interesting discoveries have been made on microorganisms associated with insects or protozoa. Unfortunately, our knowledge about the microbiology of other invertebrates like microscopic metazoa is still extremely scarce. Here, we present our first explorations of the role of microorganisms for species from the poly‐extremophile microscopic metazoan phylum Tardigrada. Tardigrades (water bears) are polyextremophilic, cosmopolitan, microscopic eukaroytic metazoans that are able to survive different types of extreme conditions, ranging from extreme temperature differences (‐273 to ~100 C), 1,000 times more ionizing radiation than most other animals such as humans, and complete desiccation for long periods, including space vacuum. Tardigrades are one of the animals on our planet that are capable of reversibly suspending their metabolism and entering into a state of cryptobiosis, where metabolism has been lowered to immeasurable levels at water content below 1 % of the normal hydration. Due to these traits, they belong to one of the few animals on our planet that can survive some of the extreme conditions found in outer space, and may thus serve as important model organisms to explore survival capabilities of organisms from planet Earth in foreign ecosystems and in outer space. Although the biology and the survival strategies of tardigrades have been studied in greater detail, our knowledge about the endogenous microbiology of tardigrades and its potential role for its survival is rather scarce. However, we anticipate that a combined research on the biology in general and the microbiology of tardigrades may add interesting insights into possible synergistic survival strategies of tardigrades. Another intriguing question to resolve is if and which types of prokaryotes in tardigrades shipped to outer space may survive, and if synergistic interactions may take place that will influence the survival possibilities of both tardigrades and the prokaryotes in an alien ecosystem. In order to trace such fascinating, though rather speculative issues, several basic studies and method developments must be performed so that reliable tools for exploring the microbial involvement under different conditions can be used in these kinds of experiments. We aim to compare different analytical and cultivation tools to explore the microbiological composition and dynamics in tardigrades. Once these tools have been optimized, they will be employed in model systems with various tardigrade species associated with different types of organisms under different conditions. We predict that this case study on Tardigrada will not only reveal interesting insights into the survival capabilities of Tardigrada under different extreme conditions on our planet Earth as well as in outer space, it will also expand our knowledge about the microbial diversity of microscopic eukaryotes since virtually no microbiological research has been undertaken on this category of animals.

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PG2/3

INVESTIGATING THE DIVERSITY AND ECOLOGY OF EPIBIOTIC BACTERIA ON FRASASSI CAVE‐DWELLING AMPHIPODS: MOLECULAR EVIDENCE FOR HOST‐SYMBIONT SPECIFICITY

Jan Bauermeister *, Jean‐François Flot, Sharmishtha Dattagupta

Courant Research Centre Geobiology, Georg‐August‐Universität Göttingen, 37077

Göttingen, Germany. * Email: [email protected]. Phone: +49 551 3913448. Fax: +49 551 397918.

Niphargus amphipods endemic to the sulfide‐rich Frasassi caves of central Italy are symbiotic with filamentous chemoautotrophic Thiothrix bacteria growing on their exoskeletons. Recent molecular and morphological analyses have revealed that the symbiotic amphipods, previously thought to be one species, comprise instead of two distinct Niphargus species. One species comprises active swimmers, prevailing but not exclusively present in stagnant and deep lakes. The second species predominantly crawls on bacterial biofilms, sediment, and rocks in shallow streams or near cave springs. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was employed on Niphargus individuals from different Frasassi cave sites to assess diversity and distribution patterns of their associated bacterial epibionts. ARISA results suggest that the composition of the epibiotic community is not dependent on environmental conditions at the respective sampling site, but rather correlates with the Niphargus host species. In cave waters where both Niphargus species co‐exist, host behavior and microhabitat preference are presumed to be crucial impediments to interspecies symbiont transference. Future studies will examine symbiont diversity using other molecular fingerprinting techniques to complement our ARISA results, and further investigate the phylogeny of the Niphargus epibiont community using various bacterial gene sequence markers. Moreover, the installation of aquaria filled with cave sediment and synthetic cave water has enabled us to maintain distinct Niphargus populations in captivity for the purpose of future long‐term studies on epibiont function and transmission under controlled conditions.

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PG2/4

WHOLE GENOME AMPLIFICATION OF A SINGLE PORIBACTERIAL CELL: INSIGHTS INTO THE GENOME OF A MARINE SPONGE SYMBIONT

Alexander Siegl and Ute Hentschel

Julius‐von‐Sachs Institute for Biological Sciences, University of Wuerzburg

Numerous marine sponges are permanently associated with phylogenetically diverse microbial consortia. Here we present a single cell genomics approach for the functional characterization of the candidate phylum Poribacteria from the Mediterranean sponge Aplysina aerophoba. FACS‐sorted microbial cells were subjected to phi29 polymerase mediated ‘whole genome amplifications’ (WGAs). The phylogenetic origin of the amplified genomic DNA was elucidated using 16S rDNA‐PCRs. Amplicons were used for cosmid library construction and 454‐pyrosequencing. Genes associated with bacterial primary and secondary metabolism were identified and their origin unraveled. Accordingly, a specific class of polyketide synthases (Sup‐PKS) was affiliated with the Poribacteria. Moreover, a novel non ribosomal peptide synthetase (NRPS) was assigned to a sponge‐specific Chloroflexi clade. Cosmid clone sequencing allowed characterization of the genomic context of these biotechnologically relevant genes. Pyrosequencing of a single amplified genome derived from a member of the Poribacteria resulted in almost 2 Mb of genomic information about this sponge symbiont. Computational data analysis provided first insights into the poribacterial metabolism and traced common features with its’ sister phyla. Single cell genomics approaches such as has been undertaken in this study contribute to a better understanding of multi‐species interactions between uncultured bacteria and their eukaryotic hosts.

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Plant ‐ Soil

PG3/1 Root colonization by Pseudomonas sp. DSMZ 13134 and effect as biological plant growth

stimulant in barley

Katharina Buddrus‐Schiemann, Michael Schmid, Anton Hartmann

Helmholtz Zentrum München, German Research Center for Environmental Health,

Department Microbe‐Plant Interactions, Ingolstaedter Landstrasse 1, D‐85764 Neuherberg, Germany

Over the last few decades, the ability of rhizosphere bacteria to promote plant growth has been considered to be of scientific, ecological and economic interest. The properties and mechanisms of interaction of these root‐colonizing bacteria have been extensively investigated, and plant protection agents that are based on these bacterial strains have been developed for agricultural applications. In the present study, the effect of the commercially available plant protection agent Proradix®, that contains Pseudomonas sp. DSMZ 13134, was examined. Using the fluorescence in situ hybridization (FISH) method with specific oligonucleotide probes and gfp‐tagging in combination with confocal laser scanning microscopy, an effective colonization of barley roots was demonstrated. We could find the Pseudomonas strain in all parts of the root especially in the root hair zone on the surface of root hairs. Beneficial plant growth promoting effects could be shown in greenhouse and field experiments. We detected higher crop yields up to 19% for barley plants treated with Proradix® in field experiments. The dry weight of shoots and the number of ears were about 44% higher than in the control in greenhouse experiments with light and nutrient stress.

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PG3/2

UPTAKE AND TRANSPORT OF N‐ACETYL‐HOMOSERINE LACTONES IN BARLEY

Tina Riedel1, Sandor Forczek2, Miroslav Matucha2, Peter Schröder1

1Department Microbe‐Plant‐Interactions, Helmholtz Zentrum Muenchen, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany

2Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Videnska 1083, 14220‐Prague 4, Czech Republic

Over the last two decades it has become apparent that bacteria are able to communicate with each other via a process named Quorum sensing (QS) and thereby orchestrate bacterial gene expression. QS is also involved in mediating the interaction between different bacterial species and between bacteria and eukaryotic organisms. N‐acetyl‐homoserine lactones (AHLs) are the major signalling molecules in QS of Gram‐negative bacteria and it has been shown that these bacterial signalling molecules are able to induce responses in plants including systemic resistance. However transport and translocation into plants remains almost unknown. We used tritium labelled C8‐and C10‐homoserine lactone (HSL) to analyze the uptake and translocation in barley (Hordeum vulgare L. cv. “Barke”) plants within the first 24h after treatment as well as the inhibition of C8‐HSL transport in barley roots. Additionally we visualized the AHL transport in the central cylinder in cross sections of maize roots via autoradiography. Despite the fact that the majority of AHLs remains attached to the outer root surface, the plant reacts to the minute concentrations of AHL incorporated with changes in the activity of detoxification enzymes.

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PG3/3

Evidence for a plant associated natural habitat of Cronobacter spp.

Michael Schmid1, C. Iversen2, I. Gontia3, R. Stephan2, A. Hofmann1, A. Hartmann1, B. Jha3, L. Eberl4, K. Riedel4, A. Lehner2

1 Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Department Microbe‐Plant Interactions, Germany

2 Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Switzerland 3 Discipline of Marine Biotechnology and Ecology, Central Salt & Marine Chemicals Research

Institute, Gujarat, India 4 Department of Microbiology, Institute of Plant Biology, University of Zurich, Switzerland

Background: Members of the genus Cronobacter are responsible for cases of meningitis with high fatality rates in neonates and necrotizing enterocolitis in infants. Some physiological features, such as the production of a yellow pigment, the formation of a gum‐like extracellular polysaccharide as well as the ability to resist desiccation during long dry periods suggest an environmental origin for these organisms. However, to date, the natural habitat of Cronobacter spp. is still unknown. Objectives: Two Cronobacter sakazakii affiliated isolates from plant roots should be characterized. Furthermore, it was the aim to screen members of the genus Cronobacter from clinical and plant origin upon their root colonization behavior. Methods: Production of siderophores, IAA production and mineral phosphate solubilizing activity was determined. The colonization behavior was analyzed by applying GFP tagged strains or fluorescent in situ hybridization using fluorescently labeled oligonucleotide probes in combination with confocal laser scanning microscopy. Results: Two bacterial strains originally isolated from plant roots were designated as C. sakazakii strains after comparative sequence analysis of their 16S‐rRNA. All investigated strains show several key features often found in plant‐associated and rhizosphere microorganisms. All of the strains solubilized mineral phosphate and produced IAA. Siderophore production was observed for all except one strain. The capability of the organisms to endophytically colonize tomato and maize root hairs was demonstrated for several strains by confocal microscopy. Conclusions: The results from our study provide strong evidence that plants are the natural habitat of Cronobacter spp.

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PG3/4

HALOTOLERANT PGPR FROM SALICORNIA BRACHIATA, AN EXTREME HALOPHYTE

Iti Gontia1, Bhavanath Jha1, Michael Schmid2, and Anton Hartmann2

1 Discipline of Marine Biotechnology and Ecology, Central Salt & Marine Chemicals Research Institute (Council of Scientific and Industrial Research),

G. B. Marg, Bhavnagar‐364002, Gujarat, India 2 Helmholtz Zentrum München, German Research Center for Environmental Health,

Department Microbe‐Plant Interactions, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany

Thirteen diazotrophic (N2 ‐ fixing) bacteria were isolated from Salicornia brachiata, an extreme halophyte growing in coastal marshes of Gujarat, India. They form growth pellicles in semisolid nitrogen free medium Nfb containing NaCl up to 4%. However, in NB medium they could grow in the presence of up to 20% NaCl. The isolates produce the auxin indole acetic acid (IAA) and siderophores, utilize ACC as sole source of nitrogen and show ACC deaminase enzyme activity. To demonstrate that the isolates were diazotrophs, nifH gene was amplified using PolF and PolR primers and the amplification products were sequenced. ACC deaminase gene was also amplified to complement the ACC deaminase enzyme activity of the isolates. Two of the isolates as well as one diazotrophic PGPR reference strain were used for the inoculation of axenically grown Salicornia seedlings under different concentration of NaCl (0‐0.5 M) and plant growth promotion was observed. Based on 16S rRNA gene homology, the isolates were identified as Pseudomonas putida, Rhizobium radiobacter, Zhihengliuella spp., Mesorhizobium sp., Brachybacterium sp. Vibrio aglinolyticus, Brevibacterium casei, Cronobacter sakazakii, Halomonas sp. Out of 13 isolates, three are suggested as new bacterial species.

20

PG3/5 TRANSCRIPTIONAL RESPONSES OF BRADYRHIZOBIUM JAPONICUM TO ENVIRONMENTAL

CHANGES

Kathrin Langa, Mandy Wenzela, Gabriella Pessib, and Michael Göttferta

aInstitute of Genetics, TU Dresden, Dresden; bInstitute of Microbiology, ETHZ, Zürich In the rhizosphere, rhizobia compete with other bacteria for resources. They also have to withstand changing environmental conditions. Rhizobial strains differ in their ability to cope with extreme pH values, high salt concentrations or an elevated temperature. We are studying the effect of these parameters on the transcriptome of B. japonicum, the symbiont of soybean. In addition to the mentioned parameters, the transcriptome is also influenced by flavonoids released by the plants. Genistein, a soybean signal, activates the LysR‐type regulator NodD1 and the two‐component regulatory system NodVW. Both systems are required for efficient expression of nodulation genes. Within the wild type, about 100 genes are inducible by genistein. The majority of these genes is not preceded by known promoter elements. Our data indicate that genistein has a much broader function than mere induction of nod genes and so far uncharacterised regulators are involved in genistein‐dependent responses.

21

PG3/6

SALIX CAPREA RHIZOBACTERIA AND ENDOPHYTES WITH POTENTIAL TO ENHANCE EFFECTIVENESS OF HEAVY METAL PHYTOEXTRACTION FROM SOIL

K. Fallmann1,2, M. Kuffner1, M. Puschenreiter2, G. Wieshammer2, S. dos Reis2

and A. Sessitsch1 1AIT Austrian Institute of Technology GmbH, Bioresources Unit, Seibersdorf, Austria;

2 University of Natural Resources and Applied Life Sciences, Vienna, Austria

Willow (Salix caprea) accumulates Zn and Cd from polluted soils and has application potential for inexpensive in situ soil remediation. To study the role of bacteria for heavy metal uptake by the plant, isolates were collected and the phylogenetic diversity of culturable rhizobacteria and endophytes was determined. A soil extraction experiment on the effect of culture filtrates of selected bacteria on Zn and Cd mobilization was conducted. A mobilizing strain and one with immobilizing effect were inoculated on S. caprea plantlets to study their influence on heavy metal accumulation. Additionally, the isolates were tested for competitive colonization of plants in sterilized soil.

Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes/Chlorobi group accounted for over 90 % of isolated rhizobacterial taxa, whereas endophytes belonged mostly to Alphaproteobacteria (over 60 %) and Actinobacteria. Heavy metal mobilizing, immobilizing and strains without effect were found. The plant‐inoculated mobilizing strain increased Zn and Cd concentration in leaves, the immobilizing strain reduced metal concentrations in roots. In the competitive plant colonization experiment, ten percent of the isolates were detected in plants after five weeks. The results support the application potential of plant‐associated bacteria to improve the effectiveness of the heavy metal phytoextraction process and accelerate soil remediation.

22

PG3/7

3‐Methylarginine, a toxin from the symbiotic epiphyte Pseudomonas syringae pv. syringae 22d/93

Janine Hofmann1, Sascha D. Braun2, Beate Völksch2, and Dieter Spiteller1

1Max Planck Institute for Chemical Ecology, Bioorganic Chemistry, Hans‐Knöll‐Straße 8,

07745 Jena, Germany. 2Microbial Phytopathology, Friedrich Schiller University Jena, Neugasse 25, 07743 Jena,

Germany. 3‐Methyl‐L‐arginine is produced by the symbiotic epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d).[1] The rare amino acid acts as strong toxin against its relative the plant pathogen Pseudomonas syringae pv. glycinea having the potential to protect its host plant, the soybean. Feeding labelled methionine to Pss22d indicated the introduction of a methyl group by an S‐adenosylmethionine (SAM)‐dependent methyltransferase. Using mutagenesis the genes for the biosynthesis of 3‐methylarginine were identified and characterized. The two genes, a methyltransferase and an aminotransferase, were overexpressed in E. coli. The methyltransferase was successfully purified and proven to catalyse the formation of 5‐guanidino‐3‐methyl‐2‐oxo‐pentanoic acid as intermediate of the 3‐methylarginine biosynthesis (Figure 1).

Figure 1. 3‐Methylarginine biosynthesis by Pseudomonas syringae pv. syringae 22d/93: Reaction of the methyltransferase Because it was impossible to obtain the second enzyme an aminotransferase as soluble protein by standard overexpression methods in E coli, the aminotransferase was overexpressed in a newly developed Pseudomonas syringae pv. syringae expression host. The use of this mutated host strain and a newly created expression vector led to the successful purification of soluble aminotransferase, the second enzyme of the 3‐methylarginine biosynthesis. [1] S. D. Braun, B. Völksch, J. Nüske, D. Spiteller, 3‐Methylarginine from Pseudomonas syringae pv. syringae 22d/93 suppresses the bacterial blight caused by its close relative Pseudomonas syringae pv. glycinea. Chembiochem 9, 1913‐20 (2008).

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PG3/8

STRUCTURE AND FUNCTION OF POTATO ASSOCIATED MICRORGANISMS IN THE CENTRAL ANDEAN HIGHLANDS

Stefan Pfeiffer, Branislav Nikolic and Angela Sessitsch

AIT Austrian Institute of Technology GmbH, Department of Health and Environment,

Bioresources Unit, A‐2444 Seibersdorf, Austria The Central Andean Highlands, confining Ecuador, Peru and Bolivia, are the origin of potato diversity, covering more than 3,000 varieties. Being the major source for carbohydrate since ancient times, increased demand by a growing population have reduced crop rotation and increased the pressure caused by pest and plant disease. The EU funded project VALORAM targets to elucidate the role of microorganisms naturally associated with the potato. Microorganisms play a key role in the plants nutrient cycle and also can offer benefits (e.g. pest resistance) to their host. The aim of this work is to cover the natural bacterial diversity of potato associated bacteria under different geographical (altitude, latitude) and seasonal (early growth stage, flowering period and senescence) conditions via 16S‐TRFLP analysis and quantitative PCR. Further, occurrence of genes providing beneficial functions to their host will be quantified within the different bacterial communities. These genes include acdS, which encodes for 1‐aminocyclopropane‐1‐carboxylate deaminase (provides stress reduction), phzH and phlF, which are involved in the synthesis of the antibiotics phenazine‐1‐carboxylic acid and phloroglucinol, and nifH, the marker gene involved in the synthesis of nitrogenase reductase (Nitrogen fixation). Knowledge of these functions could help to develop technologies to improve sustainability of potato cropping systems. An overview of the project and first results will be presented.

24

PG3/9

Microbial Endophytes From Warburgia ugandensis ‐ Diversity and Effects on Host Drimane Sesquiterpenes

Birgit Mitter1, Sigrid Drage2, Christina Tröls1, Franz Hadacek2, Alice Muchugi3, Ramni Jamnadass3 and Angela Sessitsch1

1AIT Austrian Institute of Technology GmbH, Bioresources Unit, A‐2444 Seibersdorf, Austria 2 Department of Chemical Ecology and Ecosystem Research, Faculty of Life Sciences,

University of Vienna, Althanstrasse 14, 1090 Vienna, Austria 3International Centre for Rural Agriculture and Forestry (ICRAF), Nairobi, Kenia

We present an interdisciplinary approach to study the function and physiology of culturable and non‐culturable endophytes colonizing Warburgia ugandensis, a tropical tree which is highly regarded as remedy of various human diseases caused by bacteria, fungi or viruses in traditional folk medicine. Phytochemical studies on W. ugandensis led to the identification of drimane sesquiterpenes with broad antibiotic activities. We expect that, simultaneously with this high plant physiological activity, associated endophytic bacteria also proliferate and exhibit broad functional diversity. Combining the information about the plant genetic background with the chemical analysis of secondary metabolites in W. ugandensis and the composition and function of microbes colonizing this tree comprehensive insight into the relationship between microbial diversity and the plant physiological phenotype is obtained. Our results reveal significant variations in the plant metabolite composition as well as distinct bacterial and fungal communities within individual trees and show that the endophytic community structure correlates the sesquiterpene composition within a plant. Screening of isolated fungal and bacterial endophytes for their sensitivity towards plant drimane sesquiterpenes revealed one fungi and three bacterial strains to be significantly more resistant than the control strains.

25

PG3/10

EFFECTS OF TRANSGENIC MAIZE ON BENEFICIAL PLANT‐MICROBE‐INTERACTIONS AND SOIL NITROGEN CYCLING

Michaela Prischl, Evelyn Hackl and Angela Sessitsch

AIT Austrian Institute of Technology GmbH, Health & Environment Department,

Bioresources Unit, A‐2444 Seibersdorf Through the use of transgenic crops expressing Bacillus thuringensis‐derived insecticidal proteins the need for conventional insecticides is reduced, providing benefits for human health and the environment. Limited understanding of soil‐plant‐microbe interactions in Bt‐crop‐systems, however, causes uncertainties regarding potential adverse effects on the ecosystem level. Thus, the present project investigates effects of the genetic modification on beneficial plant‐microbe interactions and on soil nitrogen cycling during a three year‐rotational cropping of maize and horsebean. Plants of three Bt‐maize varieties, an isogenic and three conventional maize lines were grown on two contrasting soils in a containment system. Flowering as well as milk ripe plants were harvested for the isolation of endophytic microorganisms, which were screened for plant growth promoting and pathogen suppressive capabilities. Endophytic DNA was extracted from the plants for microbial community analysis and PCR‐based analysis of genes encoding plant beneficial functions. At multiple time points, measurements of potential N turnover and soil mineral N content were done together with functional gene‐based analyses to assess potential differential effects of Bt‐ versus non Bt‐plants on soil N cycling. Project results will provide new information about soil‐plant‐microbe‐interactions in systems using genetically modified Bt‐maize as compared to isogenic or conventional maize lines.

26

PG3/11

RISKS AND RECOMMENDATIONS REGARDING HUMAN PATHOGENS IN ORGANIC VEGETABLE PRODUCTION CHAINS (PATHORGANIC)

Fenzl C1), Hackl E1), Brankatschk K2), Jäderlund L3), Jensen A.4), Hofmann A.5), Rinnofner T.6), Koller M.7), Friedel J. 6), Duffy B2), Arthurson V. 3), Wyss G. 7), Schmid M. 5), Baggesen D. 4)

and Sessitsch, A1)

1) AIT Austrian Institute of Technology GmbH, Health & Environment Department, Bioresources Unit, A‐2444 Seibersdorf, Austria

2) Agroscope Changins‐Wädenswil (ACW), Plant Protection Division 3) Swedish University of Agricultural Sciences, Dept. of Microbiology

4) Department of Microbiology and Risk Assessment, The National Food Institute, The Technical University of Denmark

5) Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Department Microbe‐Plant‐Interactions

6) BOKU ‐ University of Natural Resources and Applied Life Sciences, Department of Sustainable Agricultural Systems

7) Forschungsinstitut für Biologischen Landbau (FiBL) A continuous rise in the number of outbreaks of disease associated with the consumption of vegetables has been observed during the last few decades. PathOrganic addresses the quality and safety of organically produced vegetables. Multiple factors influence microbial contamination of produce, including the use of animal manure as a fertilizer. In this study, manure samples were collected from organic farms in Austria, Germany, Denmark, Sweden and Switzerland and were screened for the presence of five human pathogens most often linked to produce‐associated outbreaks: Salmonella enterica, Campylobacter ssp., Listeria monocytogenes, Staphylococcus aureus and E.coli. Of the manure samples collected in the various countries, between 38 and 70% proved positive for Campylobacter ssp. and up to 24% contained Staphylococcus aureus, while Salmonella enterica was less frequently detected. In addition, Shiga‐toxin producing E.coli virulence genes were identified in 21 to 68% of manure samples from the individual countries. In the following, produce grown on farms using contaminated manure was sampled and surveyed for the presence of the food pathogens. Staphylococcus occurred most frequently, with up to 20% positive samples. With results varying from country to country, Salmonella‐positive samples were found in 1‐15% of produce, Listeria ranged from 0 to 12%, and up to 2% of the samples proved positive for Campylobacter. Shiga‐toxin producing E.coli virulence genes were found in up to 20% of produce.

27

PG3/12

METAGENOMIC ANALYSIS OF 1‐AMINOCYCLOPROPANE‐1‐CARBOXYlATE (ACC) DEAMINASE GENES AMONG POTATO ENDOPHYTES

Branislav Nikolic1, Helmut Schwab2, Angela Sessitsch1

1AIT Austrian Institute of Technology, Bioresources Unit, Health & Environment Department, 2444 Seibersdorf, Austria

2 Technische Universität Graz, Institut für Molekulare Biotechnologie, 8010 Graz, Austria E‐mail: [email protected]

Bacterial endophytes are mostly soil‐borne, plant‐associated bacteria that are able to penetrate plants and colonize intercellular spaces and vascular tissues, where they reside at least part of their life without causing any immediate negative effects or forming any organized symbiotic structures. It has been frequently reported that many endophytes have beneficial effects on their host plant such as plant growth promotion or bio‐control of pathogens. Many of endophytes are expressing 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase that is hydrolysing ACC, a precursor in the synthesis of ethylene in plants. Lowering of ethylene commonly promotes root elongation, plant growth and resistance to various biotic and abiotic stresses. Metagenomic approaches which rely on the isolation of the entire bacterial genome of a habitat of interest combined with subsequent cloning and analysis are gaining importance in resolving the ecology and functions of yet uncultured or hardly cultivable bacteria. We constructed metagenomic libraries from potato endophytes and screened for ACC deaminase activity. Detailed construction procedures and screening results are shown, observed problems and alternative approaches are considered.

28

PG3/13

HYDROCARBON DEGRADATION AND PLANT COLONIZATION OF SELECTED BACTERIAL STRAINS ISOLATED FROM ITALIAN RYEGRASS AND BIRDSFOOT TREFOIL

Sohail Yousaf, Katrin Ripka, Verania Andria, Thomas Reichenauer, Muhammad Afzal

and Angela Sessitsch

AIT Austrian Institute of Technology GmbH, Health & Environment Department, Bioresources Unit, A‐2444 Seibersdorf, Austria

Microbial inoculation of plants with degrading strains is a promising approach in phytoremediation‐based clean‐up of petroleum polluted soils. In addition to high degradation activity high persistence and efficient colonization is a pre‐requisite for benefiting from microbial inoculation. In this study the degradation potential and plant colonization capacity of four alkane degrading strains (ITSI10, ITRI15, ITRH76, and BTRH79) in combination with Birdsfoot trefoil and Italian ryegrass was evaluated. Contaminated soil was prepared by spiking agricultural soil with 10 g diesel fuel kg‐1 soil. Soils, i.e. sterilized and non‐sterilized, with and without compost treatment were used. Based on an earlier study on screening plants for phytoremediation of hydrocarbons, Italian ryegrass (Lolium multiflorum var. Taurus) and Birdsfoot trefoil (Lotus corniculatus var. Leo) were selected for their tolerance and rapid growth response and inoculated with the strains listed above individually. Planted, but uninoculated as well as non‐vegetated treatments served as controls. Hydrocarbon degradation (up to 57 %) was observed in all inoculated treatments of vegetated and unvegetated samples. Treatments with compost showed better degradation than without compost. IT + compost + BTRH79 showed highest degradation (57 %) while BT + compost + ITSI10 being the second best treatment (48 %). Colonization analysis showed that the inoculant strains were successfully colonizing the plants both in sterile and non‐sterile soils. In sterilized soil inoculated strains colonized 80‐100 % and in non‐sterilized soil the colonization was in the range of 20 to 90 %.

29


Plant ‐ Soil; Water ‐ Waste

PG4/1

DNA BARCODING AND SEQUENCE BASED IN‐FIELD SPECIES DETECTION OF ARBUSCULAR MYCORRHIZAL FUNGI

Manuela Krüger, Herbert Stockinger, Arthur Schüßler

Ludwig‐Maximilians University Munich, Department Biology, Genetics,

Großhaderner Str. 4, 82152 Martinsried Arbuscular mycorrhiza (AM) is a widespread symbiosis between AM fungi (AMF) and >80% of vascular plants. AMF provide inorganic nutrients to their plant host and achieve carbohydrates in exchange. Their enormous ecological and economical role is obvious, but still few is known about their functional traits. A basic problem is their detection and identification in the field. Molecular characterisation was established, mostly based on the rRNA genes or the ITS rDNA region. For species‐level resolution the widely used SSU rDNA is not suitable. We developed new primers, which specifically amplify a 1.8 kb fragment covering the 3’ SSU, the ITS region and a part of the LSU rDNA region including the D1 and D2 domain. We successfully used them to DNA barcode AMF, and to detect AMF in the field with species‐level resolution. We also analysed different shorter, potential barcoding regions within this fragment. The ITS region, most likely becoming the official DNA barcode for Fungi, or the LSU‐D2 region alone cannot resolve all AMF species. Therefore, we recommend to use the complete 1.8 kb fragment noted above, or e.g. for 454 Titanium sequencing, the combination of the ITS2 and the LSU D2 region to detect AMF species.

30

PG4/2 MOLECULAR SIGNALLING IN THE TRIPARTITE SYMBIOSIS OF PIRIFORMOSPORA INDICA –

RHIZOBIUM RADIOBACTER‐BARLEY Li, D.1, Sharma, M.2, Zuccaro, A.2, Fekete, A.3, Schmid, M.1, Rothballer, M.1, Schmitt‐Kopplin,

P.3, Kogel, K.‐H.2, and Anton Hartmann1

Helmholtz Zentrum München, German Research Center for Environmental Health, 1Department Microbe‐Plant Interactions, 3Institute of Ecological Chemistry, D‐85764

Neuherberg / Munich, Germany; 2Justus‐Liebig University Giessen, Institute of Phytopathology and Applied Zoology, Giessen, Germany

Members of the fungal order Sebacinales, the most basal basidiomycota group with members forming mycorrhiza with orchids and Ericaceae, also have abilities of growth promotion and induction of systemic resistance in a variety of plants. Piriformospora indica, originally isolated from desert plants in the Tar desert in India, also belongs to this fungal order and is able to form mycorrhiza‐like plant growth promoting symbioses with many plants. A close examination of Sebacinales cultures, which showed no hints of being contaminated with bacteria, resulted in the demonstration of the presence of endofungal bacteria when using phylogenetic probes and fluorescence in situ hybridization (FISH) analysis. For example, Paenibacillus sp. was demonstrated within hyphae of Sebacina vermifera MAFF 305838, while the Gram‐negative bacterium Rhizobium radiobacter was identified as endofungal bacterium in P. indica. Until now, P. indica could not be cured from the bacterium. P. indica colonizes epidermal roots cells, leading to cell death in the invaded cortical cells. Plant growth promotion and an increase in pathogen resistance against powdery mildew was demonstrated in many plants – e.g. in barley. Most interestingly, the R. radiobacter isolates had similar effects on barley plants as the fungus (including the endofungal bacterium) In search for signalling candidates possibly involved in plant growth promotion, biosynthetic genes of indole acetic acid was demonstrated in R. radiobacter as well as in the P. indica genome. Most remarkably, the synthesis of bacterial quorum sensing compounds of the N‐acylhomoserine lactones (AHLs), like oxo‐ and hydroxy‐C8‐, C10‐ and C12‐HSL, were identified using FTICR‐mass spectrometry. AHLs have recently been demonstrated to be able to modulate plant growth or even increase pathogen resistance in different plants. In addition, Rhizobium radiobacter produced HSL‐compounds using the plant‐derived compound coumaroylic acid as side chain. We therefore hypothesize that the endofungal bacteria and their signalling compounds are synergistically involved in the interplay of the partners in this triple symbiosis of plant, the fungus P. indica and the bacterium R. radiobacter.

31

PG4/3

Symbiotic based cultivation for improved cultivation strategy of novel species within the phylum Chloroflexi

N. M. Lee1; D. B. Meisinger1, K. M. Ritalahti2, S. Spring3, M. Schmid4, F. E. Löffler2

1Microbiology, TUM, Freising, Germany; 2Environmental microbiology and engineering, GaTech, GA, USA; 3DSMZ, Braunschweig, Germany; 4Helmholtz Zentrum München,

Department Plant Microbe Interactions, Neuherberg, Germany,

With the development of the molecular techniques in microbiology, it became evident that the majority (around 1 %) of the bacteria in the environment are not yet culturable despite large cultivation and screening efforts. Several recent studies have shown that the enrichment strategy of novel bacteria can be improved by co‐cultivation probably due to beneficial symbiotic interactions. Here, we demonstrate how this approach helped to isolate or improve cultivation of several novel species and genera affiliated to the phylum Chloroflexi. The Chloroflexi constitute a poorly defined, deeply branching lineage on the bacterial phylogenetic tree, but the constantly growing amount of 16S rRNA gene sequences retrieved from unculturable organisms in ecosystems from all parts of the world (extreme, non‐extreme, pristine, contaminated) affiliated to this phylum suggest that an unexplored diversity exists. The phylum harbors both organisms relevant to the study of early evolution of microbial life (e.g. one of the first anoxygenic phototrophs) in extreme and non‐extreme environments, as well as bacteria of importance for environmental biotechnological applications such as bioremediation. Here, we describe a collaborative effort to explore the biodiversity, ecology and culturability of Chloroflexi affilated bacteria. We screened different environments, including wastewater treatment plants, soils, chemo‐autotrophic pristine subsurface areas, and contaminated groundwater systems, on geographically distinct locations. To date, we succeeded in retrieving ~ 300 novel Chloroflexi‐affiliated 16S rRNA gene sequences, as well as isolating nearly 30 novel Chloroflexi isolates. These isolates were characterized by a polyphasic approach and found to constitute several novel genera, affiliated with classes I, II, III and VI within Chloroflexi. Interestingly, many of the novel isolates, similar to previous observations on other Choroflexi affiliated strains from 3 of the 6 currently recognized subgroups within Chloroflexi, showed increased culturability when cultivated together with other specimens. We are currently evaluating possible mechanisms of the symbiotic interactions and whether these can be used to increase the culturability of other so far unculturable species affiliated to Chloroflexi.

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PG4/4

BIOLOGICAL ROLE OF SECONDARY METABOLITES FROM MARINE MICROORGANISMS Heike I. Baumann, Jutta Wiese, Andrea Gärtner, Franz Goecke, Herwig Heindl, Inga Kajahn,

Katrin Kleinschmidt, Antje Labes, Kerstin Nagel, Sven Neulinger, Birgit Ohlendorf, Rolf Schmaljohann, Imke Schneemann, Dirk Schulz, Tim Staufenberger, Rüdiger Stöhr, Vera Thiel,

Heidi Zinecker and Johannes F. Imhoff

Kieler Wirkstoff‐Zentrum (KiWiZ) at IFM‐GEOMAR, Am Kiel‐Kanal 44, D‐24106 Kiel IFM‐GEOMAR at the University Kiel, Düsternbrooker Weg 20, D‐24105 Kiel

The marine environment is still rarely investigated with respect to biological interactions mediated by microbial metabolites. The Kieler Wirkstoff‐Zentrum KiWiZ (Center of Marine Natural Products) in Kiel focuses on natural products from marine microorganisms. The KiWiZ combines microbiological, biotechnological, genetic, and chemical expertise to identify natural products concerning their ecological function like symbiotic/defensive interactions as well as their application areas. We will present different projects which deal with bacteria‐sponge interactions, bacteria‐macroalgae interactions and bacteria‐bacteria interactions. In addition, new metabolites produced by sponge‐associated fungi from marine habitats and their biological activities will be demonstrated.

33

PG4/5

MOLECULAR AND PHYSIOLOGICAL CHARACTERIZATION OF THE CLOSE BACTERIAL SYMBIOSIS IN PHOTOTOPHIC CONSORTIA

Johannes Müller1, Roland Wenter1, Dörte Dibbern1, Veronika Reisinger2, Matthias Plöscher2,

Lutz Eichacker2 and Jörg Overmann1

1Bereich Mikrobiologie, Department Biologie Ι, Ludwig‐Maximilians‐Universität München, Großhadernerstr. 2‐4, 82152 Planegg/Marinsried2Bereich Botanik, Department Biologie Ι, Ludwig‐Maximilians‐Universität München, Großhadernerstr. 2‐4, 82152 Planegg/Marinsried The phototrophic consortium "Chlorochromatium aggregatum" represents the most highly developed symbiosis among bacteria known. It consists of green sulfur bacterial epibionts surrounding a central, motile Betaproteobacterium. "C. aggregatum" represents the first cultivable model system to dissect the molecular basis of symbiotic interaction between non‐related prokaryotes. To identify proteins involved in interspecies interaction, membrane proteins were analyzed after cross‐linking. Symbiosis‐dependent regulation of the epibiont transcriptome and proteome was studied using prokaryotic cDNA subtractive hybridization, whole‐transcriptome sequencing and two‐dimensional differential gel electrophoresis. The cross‐linking experiments imply that a hemagglutinin‐like protein and a branched‐chain amino acid ABC‐transporter are directly involved in the cell‐cell interaction. A total of 354 epibiont genes were differentially expressed either in the symbiotic or free‐living state indicating that the interaction involves a distinct regulation. Since most genes coding for metabolic functions are also present in free‐living relatives, the ancestor of the epibiont might have been preadapted to symbiotic interactions. By 16S rRNA based specific magnetic capture, the kinetics of H14CO3

‐ incorporation into the epibiont and central bacterium were monitored simultaneously. Almost synchronous 14C labeling of epibiont and central bacterium rRNA demonstrated a rapid transfer of low molecular photosynthesis products to the heterotrophic partner.

34

PG4/6

ECOPHYSIOLOGY AND CELL‐CELL‐INTERACTION OF A NOVEL MULTICELLULAR MAGNETOTACTIC PROKARYOTE FROM NORTH SEA SEDIMENTS

Roland Wenter1, Gerhard Wanner2, Dirk Schüler1 and Jörg Overmann1

1Bereich Mikrobiologie, Department Biologie Ι, Ludwig‐Maximilians‐Universität München, Großhadernerstr. 2‐4, 82152 Planegg/Marinsried

2Bereich Botanik, Department Biologie Ι, Ludwig‐Maximilians‐Universität München, Großhadernerstr. 2‐4, 82152 Planegg/Marinsried

Multicellular magnetotactic prokaryotes (MMPs) represent highly organized, spherical and motile aggregates of 10‐40 bacterial cells, each with its own magnetosomes and flagellation. The entire aggregate orients itself in a magnetic field and exhibits magnetotaxis. Cells of MMPs recovered from coastal tidal sand flats of the North Sea were found to contain bullet‐shaped magnetosomes which were aligned in several parallel chains, all oriented in the same direction across the entire aggregate. This particular arrangement of magnetosomes has not been observed previously and indicates a close signal exchange between the cells of one aggregate. The 16S rRNA gene sequence of MMPs from the North Sea exhibited phylogenetic distances of more than 4% to all other bacteria. Fluorescence in situ hybridization demonstrated that the entire population of MMPs and all individual cells of the same multicellular aggregate belonged to the same phylotype. Genes for dissimilatory sulfite reductase (dsrAB) and dissimilatory adenosine‐5´‐phosphate reductase (aprA) were detected in magnetotactically purified MMP samples, suggesting that the MMPs are capable of sulfate reduction. Chemotaxis assays with 41 test substances yielded strong responses towards acetate and propionate, which are typical growth substrates for sulfate‐reducing bacteria. MMPs may serve as future model system for studying bacterial multicellularity and interaction.

35

PG4/7

A ROLE FOR OLIGOCHAETE‐ENDOSYMBIOTIC MICROBES IN SULPHUR‐CYCLING IN CONTAMINATED AQUIFERS?

Giovanni Pilloni, Tanja Riedel, Kathrin Euringer, Claudia Kellermann and Tillmann Lueders

Institute of Groundwater Ecology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH) Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.

giovanni.pilloni@helmholtz‐muenchen.de Marine gutless Oligochaetes are well known to harbor characteristic communities of chemoautotrophic endosymbiotic Bacteria, via whom they gain energy and carbon while shuttling between different redox zones of marine sediments. These symbioses include specific sulfate‐reducing and sulfide‐oxidizing endosymbionts, which acquire organic and inorganic carbon for the host, and maintain an endosymbiotic sulfur cycle within the worm. Thus, diffusive limitations and spatial separation of available electron donors and acceptors can be overcome. Also in contaminated aquifers, degradation processes are often limited by a spatial separation of electron donors (contaminants, sulphide) and thermodynamically attractive electron acceptors (oxygen, nitrate). Thus, also in this environment, a mobile worm shuttling chemolithoautotrophic endosymbionts between reduced and oxidized zones may be hypothesized as a possible multitrophic entity. Here, we provide first circumstantial evidence for the existence of such symbioses This concept is based on three independent lines of evidence that have appeared in our recent depth‐resolved gene‐based characterization of microbial community distribution in a hydrocarbon‐contaminated aquifer in Düsseldorf, Germany. Genetic signatures of the functional genes specific for sulfate reduction (dsrB) and for CO2 fixation (Rubisco), as well as eukaryotic 18S rRNA genes detected on site, amongst others, all recovered sequence types closely related to Olavius algarvensis or its known bacterial endosymbiotic partners. This intriguing observation evoked our working hypothesis that such microbe‐eukaryote symbioses may be extant at redox gradients not only in marine, but also in groundwater environments. Here, we discuss different aspects supporting or contradicting this hypothesis, and future research objectives that may substantiate these so‐far overlooked elements of groundwater ecology.

36

PG4/8

Homogeneous inoculation vs. microbial hot spots of isolated strain and microbial community: What is the most promising approach in remediating soils

contaminated with organic chemicals?

A. Krug1, F. Wang1, U. Dörfler1, D. Fischer2, M. Schmid2, J.C. Munch1, R. Schroll1

1 Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Institute of Soil Ecology, 85764 Neuherberg, Germany

2 Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Department Microbe‐Plant Interactions, 85764 Neuherberg, Germany

We artificially loaded different type of agricultural soils with some model 14C‐labelled chemicals, and we inoculated such soils with different microbial communities as well as isolated strains to enhance the mineralization of such chemicals. Inocula were introduced by different approaches: (i) soil inocula, (ii) application of isolated strain as well as microbial community via media, (iii) isolated strain as well as microbial community attached to a carrier material. Most of the inoculation experiments were conducted in laboratory but we also tested one of these approaches under real environmental conditions in lysimeters and we could show that the approach was successful. We already could show that inoculating soils with microbial communities attached on a specific carrier material shows the highest mineralization effectiveness and also the highest sustainability. Microbes attached on clay particles preserved their function over a long time period even if the specific microbial substrate was already degraded or at least not detectable any more. Additionally we already could show that in specific cases some soil parameters might reduce the effectiveness of such an approach. Results on isoproturon as a model for phenylurea‐herbicides and 1,2,4‐trichlorobenzene as an example for an industrially used chemical as well as the corresponding chemicals` degrading microbial communities and isolated strain will be presented.

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