Das Projekt "Analyse des in-situ Verhaltens von gentechnisch behandelten Bakterien in einem standardisierten Mikrokosmos" wird vom Umweltbundesamt gefördert und von Gesellschaft für Biotechnologische Forschung mbH durchgeführt. Achievements: Activated sludge microcosms simulated the level of aeration. Nutrient makeup and microbial community structure associated with activated sludge reactors. Soil microcosms were initially sterilised, but maintained many of the physicochemical characteristics of soil. The fate and behaviour of the genetically engineered microorganisms (GEMs) were consistent within microcosms and allowed for comparisons to be made between microcosm types. Polyclonal and monoclonal antibodies were specific for the GEMs when tested against closely related Pseudomonads and bacteria isolated from the microcosms. Nucleic acid probes were specific for recombinant deoxyribonucleic acid (DNA), did not hybridise to closely related microorganisms and isolated bacteria and were used in colony hybridisation procedures to detect plasmid DNA in putative transconjugant bacteria. GEMs maintained population levels of 10(4) bacteria/ml sludge. In soil microcosms, the density of GEMs varied depending on physicochemical properties. The GEMs degraded substituted aromatic compounds present in both microcosms. Recombinant DNA was stable in GEMs added to the microcosms both in the absence and presence of the aromatic compounds which they specifically degrade P. sp. strain FR1(pFRC20P) contains recombinant DNA necessary for the degradation of alkylbenzoates on both the chromosome and plasmid, pFRC20P. Transfer of this DNA was not detected in any microcosm. The plasmid was mobilised in vitro by helper plasmids at frequencies of 10(-5) transconjugants/recipient. P. putida (pWWO-EB62) has the modified catabolic pathway for ethylbenzoate present on the plasmid. Transconjugants arising by transfer of plasmid, pWWO-EB62, to recipient bacteria were observed in activated sludge microcosms at densities of 1000 bacteria/ml and in soil microcosms. The frequency of in vitro conjugative transfer of pWW0-EB62 to recipient bacteria was 1 to 0.1 transconjugants per donor cell. Conjugative transfer of pWWO-EB62 was analysed on solid medium and continuous culture. On agar, the plasmid transfers to and is expressed in Pseudomonads group I and Escherichia coli. Other bacteria including Pseudomonads belonging to group II, III and IV did not act as recipients, either because the plasmid was not transferred or stably maintained. In continuous culture, the transfer rate of pWWO-EB62 from P. putida KT2440 (pWWO-EB62) to P. putida UWC1, was dependent on cell doubling time, the temperature, and degree of agitation.
Das Projekt "Risikobewertung fuer das Freisetzen genmanipulierter Mikroorganismen" wird vom Umweltbundesamt gefördert und von Universität Bayreuth, Bayreuther Zentrum für Molekulare Biowissenschaften, Lehrstuhl für Genetik durchgeführt. Objective: Microorganisms deliberately released into the environment after rigorous testing are unlikely to prove a direct threat (i.e. as pathogens), and prediction of such risks is almost impossible. However, the main risk is the prospect of creating potentially hazardous new organisms following the transfer of mobile genes to native bacteria. Many bacteria can exchange genetic material, including plasmids and transposons; therefore the persistence and spread of both the organisms and genes are both important and would be affected by a variety of selective pressures. The results to be expected by this study are relevant at the methodological level as well as for its long term objectives. Techniques for sampling bacteria in the soil and for screening for the spreading of the manipulated microorganisms in the field, should be standardized. A precise evaluation of the effect of the selection pressure effect in the field of different systems should be obtained. An overall assessment of potential risks involved in the release of the respective bacteria into the environment would come from the analysis of the persistence of the inoculant strains in the field and from the evaluation of the transfer of the introduced genes to other bacteria in the soil. General Information: Aim of the research to be conducted in this research project is, - in cooperation with two other European laboratories - to monitor the persistence of genetically manipulated bacteria introduced into agricultural soils and to screen for the spread of genes carried by these micro-organisms to other members of the soil flora. Screening for the spreading of the genes will be performed by selection for the antibiotic resistance markers and by specific hybridization of DNA from the field strains with labelled probes for the introduced genes. Achievements: To investigate survival of introduced strains and their genes and to develop and assess monitoring methods, genetically marked derivatives of 2 common soil bacteria were used: Rhizobium which fixes atmospheric nitrogen in the root nodules of legumes (and has a long history) of safe and effective use as an agricultural inoculant) was used in both field and laboratory experiments; Enterobacter agglomerans which fixes nitrogen in association with cereal roots was studied in the laboratory. Strains were marked with genes conferring antibiotic resistance, either by selecting naturally occurring chromosomal mutations, or by insertion of transposon Tn5 to conjugative plasmids. In addition, native Rhizobium populations were screened for circumstantial evidence that genetic exchange occurs (over a long period) in the environment. The Tn5 marker enabled extensive monitoring of the Rhizobium inoculant, which showed significant variation in survival in field soils of the collaborating countries. The host plant was not required for its establishment.
Das Projekt "Populationsgenomik baltischer Schweinswale - Individuenspezifische genetische Populationszuordnung baltischer Schweinswale mittels hochauflösender Single Nucleotide Polymorphisms (SNPs)-Technologie" wird vom Umweltbundesamt gefördert und von Universität Potsdam, Mathematisch-Naturwissenschaftliche Fakultät, Institut für Biochemie und Biologie, Professur für Evolutionsbiologie , Spezielle Zoologie durchgeführt. Ziel dieses Projekts ist es, einen repräsentativen Datensatz von baltischen Schweinswalen individuenspezifisch genetisch zu typisieren, um - die Existenz einer separaten Schweinswalpopulation in der inneren Ostsee zu überprüfen. - Das Vorkommen dieser Population regional und saisonal zu bestimmen und - Individuen dieser Population gegen saisonal migrierende Individuen anderer Populationen abzugrenzen. Mittels RAD-tag genotyping by sequencing sollen SNPs in einigen hundert Individuen untersucht werden (je mindestens 100 aus innerer Ostsee, Beltsee/Kattegat und Nordsee/Skagerak). Falls möglich, werden Proben aus der Fortpflanzungszeit bevorzugt berücksichtigt. Weitere Individuen werden anteilig für Außengruppenvergleiche (Proben aus Island, Spanien liegen vor) sowie zur weiteren Verstärkung des Ostsee-Datensatzes verwendet (z.B. Berücksichtigung zusätzlicher Proben außerhalb der Fortpflanzungszeit zum Erkennen von migrierenden Individuen). Auf der Basis dieser SNP-Typisierung werden individuenspezifische genetische Profile erstellt. Diese werden genutzt, um Populationen zu identifizieren und die Individuen diesen Populationen zuzuordnen. Hierdurch werden folgende Fragen beantwortet werden: - Wieviele (Sub-)Populationen gibt es im Bereich Ostsee/Skagerak/Nordsee? - Wo (Zuordnung möglichst zu 50x50km ICES Quadranten) und wann (saisonale Zuordnung) sind diese Populationen anzutreffen? - Welches Ausmaß hat saisonale Migration? Inwieweit führt sie zu genetischem Austausch? Betrifft sie beide Geschlechter in gleicher Weise oder migrieren Männchen stärker?
Das Projekt "Qualitative und quantitative Analyse des konjugativen Gentransfers auf bisher nicht-kultivierbare Mikroorganismen in mikrobiellen Lebensgemeinschaften" wird vom Umweltbundesamt gefördert und von Gesellschaft für Biotechnologische Forschung mbH durchgeführt. Der konjugative Gentransfer auf Mikroorganismen in dem Belebtschlamm einer Modellklaeranlage soll umfassend analysiert werden. Dabei wird durch den Einsatz eines neuentwickelten genetischen Systems der Nachweis des Gentransfers nicht nur auf kultivierbare und sondern erstmals auch auf nicht-kultivierbare Bakterien erfolgen und gleichzeitig soll eine taxonomische Eingruppierung dieser nicht-kultivierbaren Organismen durch vergleichende rDNA-Sequenzanalysen vorgenommen werden. Der Einfluss von umweltrelevanten Stoerungen im Klaeranlagenbetrieb auf den konjugativen Genfluss ist ein weiteres Teilziel des Projektes, das in seiner Gesamtheit neue wichtige Erkenntnisse fuer die biologische Sicherheitsforschung liefern wird.