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Analyses of microbial communities at biogeochemical interfaces and their interaction with organic pollutants in soil Kornelia Smalla, Holger Heuer and Newton Gomes Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenvirologie, Mikrobiologie und biologische Sicherheit, Messeweg 11-12, D 38104 Braunschweig Objective Introduction This project aims to explore the composition Microbes are the major drivers of geochemical and biotransformation processes in soils. In concert with the inorganic and organic constituents microbes are supposed to actively Methodological Approach This project aims to explore the composition of the microflora and their horizontal gene pool in response to phenanthrene and hexadecane and varying interfaces in soil in an interdisciplinary effort. Characteristics of biogeochemical interfaces affect the composition of colonising microbial communities and the abundance and diversity of their mobile genetic elements (MGEs) Hypotheses Soils: (A) Cambisol, (B) Luvisol, (C) Chernozem Soils (A-C) + phenanthrene Soils (A-C) + hexadecane Sampling of batch experiments at t 0, 21, 62 Batch experiments Artificial soil (Cambisol) Cambisol Sampling Percolation water and 10 x 1 cm slices from the receiving layer Artificial soil + POP S R The fate and the effects of the POPs is influenced by the soil type because the structural and functional diversity of the microbial community is dependent on the soil type each consisting of soil particle fractions and interfaces Cambisol + POP concert with the inorganic and organic constituents microbes are supposed to actively shape the architecture of the soil matrix by formation and restructuring of aggregates. The physical, chemical and biological heterogeneity of the interfaces in soil is the basis of the enormous extent of structural and functional microbial diversity typically found in soil. Despite its important role microbial life in soil remained largely a “black box” until recently because the tools required to study the structural and functional diversity of soil microbes at the various different interfaces in soil and their interaction with POPs were missing. In collaboration with Kai Totsche (FSU) Pre-work done (tool box development) DNA/RNA acid extraction DGGE of bacterial and fungal communities (16S/18S) Functional gene arrays DGGE of PCR-amplified degradative genes qPCR of degradative genes Capture of MGEs Transfer frequencies determined Molecular characterisation of the MGEs captured Chemical analysis M. Spiteller R soil particle fractions and interfaces Different interfaces are formed depending on the mineral and organic matter composition of the soils DNA/RNA extraction Recovery of soil interfaces Biparental mating Exogenous isolation of MGEs from bacteria colonising different soil interfaces Recipient, rif r , gfp + (P. putida Cupriavidus necator) Expression of degradative genes in soil bacteria followed by RT-PCR from mRNA and subsequent Southern blot hybridisation References Two additional populations appeared at 21 dpi DGGE community fingerprints to identify changes in the relative abundance in response to pollution A: 99% Burkholderia sp. RP007 B: 98.85% similarity to Nocardia vinacea ndo-DGGE community fingerprints reveal changes in the relative abundance of degradative genes in response to POP pollution Control soil No naphthalene Soil + naphthalene (2mg/g) 0 3 21 0 3 21 Bacterial communities (Donor) with degradative genes on MGEs Transconjugants, gfp + , rif r , degradative capacity (I) soil samples amended with naphthalene (II) soil samples amended with naphthalene and P. putida KT2442 (pNF142) inoculated GOMES, N. C. M., COSTA, R., SMALLA, K. 2004. Rapid simultaneous extraction of DNA and RNA from bulk and rhizosphere soil. In: G. A. Kowalchuk, F. J. de Bruijn; I. M. Head, A. D. Akkermans, J. D. van Elsas (Eds). Molecular Microbial Ecology Manual. 2nd ed. Dordrecht: Kluwer Academic Publishers, pp 159-169. GOMES, N. C. M., KOSHELEVA, I., ABRAHAM, W-R., SMALLA, K. 2005. Effects of the inoculant strain Pseudomonas putida KT2442 (pNF 142) and of naphthalene contamination on the soil bacterial community. FEMS Microbiol. Ecol. 54, 21-33. GOMES, N. C. M., BORGES, L., PARANHOS, R., PINTO, F. N., KRÖGERRECKLENFORT, E., MENDONÇA-HAGLER, L. C. S., SMALLA, K. 2007. Diversity of ndo genes in mangrove sediments exposed to different sources of PAH pollution. Appl. Environ. Microbiol. 2007 (after minor revision accepted for publication)
