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

[2] Li, J. et al (1995): J. Nat. Prod, 58(7), 1081-1086.[3] Bode HB (2009): Curr Opin Chem Biol 13(2), 224-30.[4] Reimer, D. et al (2009): ChemBioChem, 10, 1997-2001SIP013The protein phosphatase MtPP2C2 as regulator of thesymbiotic receptor-like kinase MtDMI2 in MedicagotruncatulaN. Rieger*, N. RequenaInstitute of Botany, Plant-Microbial-Interactions, Karlsruhe Institute ofTechnology (KIT), Karlsruhe, GermanyThe mutualistic arbuscular mycorrhizal (AM) symbiosis is one of theevolutionary oldest symbioses of plants, formed between fungi of theGlomerymycota and roots of the majority of vascular flowering plants. Theestablishment of the AM symbiosis requires a specific signal exchangebetween both partners. In the last years a large progress has been made inidentifying signal molecules and pathways involved. The plant receptor-likekinase DMI2 (Does not make infections 2) which is located to the plasmamembrane plays an important role in the recognition of AM fungi. DMI2 ispart of the common symbiotic signaling pathway (SYM) shared by AMfungi and Rhizobium bacteria. Using the kinase domain of Medicagotruncatula DMI2 as bait in a yeast-two-hybrid assay, we could identify aprotein phosphatase typ 2C (MtPP2C2) as a DMI2 interacting partner. Thespecificity of this interaction was confirmed in yeast by testing other kinasesas well as another phosphatase. Bimolecular fluorescence complementationassays in Nicotiana benthamiana and in vitro pull-down assays revealed alsoa specific interaction between DMI2 and PP2C2. Expression analyses inmycorrhized roots by quantitative PCR showed that MtPP2C2 expression isindependent of mycorrhization. Inactivation experiments using RNAinterference and overexpression studies are in progress to investigate the roleof PP2C2 during mycorrhiza formation. Plant PP2Cs are known asregulators of signal transduction pathways involved in growth, development,responses to hormones and abiotic stress as well as in defense responses. Inparticular, PP2C2 has been recently being shown as regulator of receptorkinases involved in pathogenic interactions. Therefore a role for MtPP2C2as regulator of the DMI2-dependent symbiotic signaling pathway inmycorrhiza is proposed.SIP014Diversity and distribution of methanogens in highertermitesJ. Nonoh*, K. Paul, A. BruneMax Planck Institute for Terrestrial Microbiology and Laboratory forMicrobiology, Philipps-University Marburg, GermanyTermites produce significant amounts of methane. Preliminary analysisindicated that in phylogenetically higher termites, which emit more methanethan lower termites, archaeal diversity and community structure is higherand more complex. While the methanogenic community in lower termitesseems to consist exclusively of Methanobacteriales colonizing the gut walland the cytoplasm of certain gut flagellates, little is still known about thediversity, location, and distribution of methanogens in the different taxa ofhigher termites. Our clonal analysis of archaeal 16S rRNA genes revealedhighest diversity in the guts of soil-feeding Cubitermes and Apicotermesspecies, with nearly all major lineages of methanogens represented; diversityof methanogens in fungus-cultivating Odontotermes and grass-feedingTrinervitermes species was lower. In addition, we recovered also a deeplybranching lineage of Euryarchaeota distantly related to uncultivatedThermoplasmatales from all the termites investigated. Presently it is notclear whether members of this group are also methanogenic. mcrA geneanalysis yielded three clusters of this functional marker of methanogenicarchaea: one affiliated with Methanobacteriales, the second withMethanomicrobiales and a third representing a deeply branching andhitherto uncultivated lineage, which is subject of further investigations.Analysis of community structure showed heterogeneous distribution ofarchaeal populations in the highly compartmentalized gut of a Cubitermesspecies, apparently reflecting the varying physic-chemical conditionsencountered in the different compartments. The highly alkaline anterior gutregions were predominantly colonized by Methanosarcinales and theposterior gut regions by Methanobacteriales, Methanomicrobiales and theThermoplasmatales related lineage. An apparently termite-specific cluster ofCrenarchaeota was found to colonize the crop and the rectum.SIP015Isolation of secreted Glomus intraradices signalsactivating Medicago truncatula mycorrhiza-specific earlyinduced genesC. Albarran*, H. Kuhn, N. RequenaInstitute of Botany, Plant-Microbial Interactions, Karlsruhe Institute ofTechnology (KIT), Karlsruhe, GermanyArbuscular mycorrhizal (AM) fungi form long-term symbiosis with roots ofmore than 80% of all land plants and are obligate biotrophs. Theestablishment and maintenance of a mutualistic symbiosis requires constantsignal exchange between both partners to avoid the host defense reactionsthat would jeopardize the association. In the AM symbiosis, is the deliveryof fungal effectors molecules, termed Myc-factors, from earliest fungal lifecycle stages the way to initiate the symbiotic program even before bothorganisms contact. Although our understanding of the molecular dialoguebetween AM fungi-host has been improved in the recent years, speciallywith the identification of the plant signal and some clues about the nature ofthe Myc-factors, still little is know about the effect in planta or the signaltransduction pathway used to decode the fungal signal.In our group, it has been recently shown that some Medicago truncatulagenes are specifically induced at early stages by diffusible signals producedby the fungus Glomus intraradices. While the gene activation is partiallytravelling through the symbiotic transduction pathway (SYM pathway) wehave shown that a second cascade is required for the activation of some ofthose early genes. This suggests that probably several Myc-factors aresecreted at the same time by the fungus. Changes in the expression pattern ofthose early-induced genes will be monitored by real time RT-PCR uponcontact with different fungal exudates in order to isolate and characterize thedifferent fungal substances. Furthermore, the use of SYM-mutant plant lineswill allow distinguishing the signalling cascade that leads to the activation ofeach gene for each compound.SIP016Generation of molecular tools for functional genomics ofbifidobacteriaM. Gleinser*, D. Zhurina, C.U. RiedelDepartment of Microbiology and Biotechnology, University of Ulm, Ulm,GermanyBifidobacteria represent an important group of the human gut microbiotaand many strains were shown to have probiotic properties such as inhibitionof pathogens, reinforcement of intestinal barrier function or antiinflammatoryeffects. However, the molecular mechanisms are largelyunknown and tools for the genetic characterization of these effects arescarce. Thus, there is a need to develop tools for functional genomics ofbifidobacteria.Here, we report the generation of a range of E. coli-Bifidobacterium shuttlevectors based on the previously published pMDY23 plasmid 1 . Weconstructed plasmids with different antibiotic resistance includingchloramphenicol (pMGC), erythromycin (pMGE), ampicillin (pMGA) andspectinomycin (pMGS). Furthermore, different promoters were cloned intothese plasmids which should allow inducible or constitutive expression ofproteins in various Bifidobacterium strains. Moreover, plasmids were shownto replicate stably over at least 100 generations in the absence of selectivepressure and hence can be used in vitro as well as in in vivo studies.To test their functionality, different proteins were cloned under the controlof various promoters. The fluorescent protein Pp1 of Pseudomonas putidawas cloned under control of bile (P bs) and starch (P st) inducible promoters inpMGS and the resulting plasmids were transformed in B. bifidum S17, B.breve S27 and B. longum/infantis E18. Our results indicate that expressionfrom P st can be induced in both E. coli and Bifidobacterium species byadding 1% starch to the growth medium as detected by fluorescentmicroscopy. pMGS containing Pp1 under the control of P st will be furthertested in vivo to assess colonization dynamics of bifidobacteria in thegastrointestinal tract of mice.Moreover, these plasmids can be used for overexpression of proteinspotentially involved in the probiotic effects of bifidobacteria. BopA, a cellsurface protein involved in adhesion of B. bifidum to intestinal epithelialcells 2 was cloned as a His-tagged fusion under control of P BAD forarabinose-inducible expression. The fusion protein was successfullyexpressed in E. coli DH5a and purified by Ni-NTA affinity chromatography.Purified protein was analyzed in adhesion experiments in competition towhole B. bifidum S17.spektrum | Tagungsband 2011