1. Institute of Soil Ecology Diversity of cbbL genes from autotrophic bacteria in differently managed agricultural soils Draženka Selesi, Susanne Stein, Isabelle Pattis, Michael Schmid and Anton Hartmann GSF National Research Centre for Environment and Health, Institute of Soil Ecology, München- Neuherberg, Germany b Introduction Autotrophic bacteria of terrestrial environments may play a significant part in the conversion of carbon dioxide into organic matter and microbial biomass and may thus contribute to the global carbon cycling. Autotrophic microorganisms accomplish this metabolism by the Calvin-Benson-Bessham cycle, in which the key enzyme ribulose-1,5-bisphosphate carboylase/oxygenase (RuBisCO) catalyzes the first, rate-limiting step. The large subunit of form I RubisCO is encoded by gene cbbL (Kusian et al., 1997) and phylogenetically subdivided in two major groups, ‘green-like’ and ‘red-like (Fig. 1). Objectives to investigate the efficiency of cbbL as a functional marker for terrestrial CO 2 -fixing bacteria to assess the diversity of ‘green-like’ and ‘red-like cbbL genes in terrestrial habitats to elucidate variations in community composition on the basis of cbbL genes in differently managed soils. Materials and Methods To gain insight into the genetic diversity of CO 2 -fixing bacteria in soil habitats we developed PCR-based assays targeting the large subunit gene cbbL of the form I RubisCO. Based on the calculation of phylogenetic relationships we designed different primer sets with strong specificity for ‘red-like’ and ‘green-like’ cbbL sequences of selected terrestrial autotrophs. Bulk genomic DNA was isolated from agricultural soils with rye crop at different long-term fertilization as well as from a soil under clover/gras cover. RFLP and phylogenetic analysis of the amplified cbbL sequences were performed. Results and Discussion Fig.1: cbbL based phylogenetic tree reflecting the affiliation of ‚green-like‘ and ‚red-like‘ sequences (modified from Watson et al., 1997) References: Kusian, B. and Bowien, B. 1997 Organization and regulation of cbb CO 2 genes in assimilation autotrophic bacteria. FEMS Microbiol. Rev. 21: 135-155 Watson, G.M.F. and Tabita, F.R. 1997 Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a molecule for phylogenetic and enzymological investigation. FEMS Microbiol. Lett. 146: 13-22. Acknowledgement: This study is supported by the Deutsche Forschungsgemeinschaft (SPP1090, Ha 1708/6). Perspectives Quantification of ‘green-like’ and ‘red-like’ cbbL genes by TaqMan-PCR Assessment of the cbbL transcript levels Effect of H 2 -treatment on the diversity of cbbL genes 800 bp M 1 2 3 4 M 1100 bp Fig. 2: 1: soil with rye crop (Halle soil) without fertilization, 2: Halle soil with animal manure, 3: Halle soil with mineral fertilizer, 4: Nitrobacter vulgaris (positive control), M: MWM Efficiency of cbbL G amplification from soil successful detection of green-like cbbL fragments from soil differences in cbbL amplification depending on the kind of fertilization were observed RFLP analysis of cbbL G amplificates low variability of the RFLP patterns low diversity of cbbL G sequences no detectable variation in pattern composition within differently managed soils Phylogenetic analysis of cbbL G cbbL G sequences amplified from the different soil samples are closely related to cbbL G of Nitrobacter vulgaris and N. winogradskyi Efficiency of cbbL R amplification from soil successful detection of red-like cbbL fragments from soil no differences in cbbL amplification depending on the kind of fertilization were observed RFLP analysis of cbbL R amplificates high variability of the RFLP patterns low diversity of cbbL R sequences in halle soil without ferilization (1), high diversity in Halle soils treated with animal manure and mineral fertilizer (2) significant variations in the composition of the cbbL- containing community in differently managed Halle soils Phylogenetic analysis of cbbL R Fig. 5: Phylogenetic tree based on ‘red-like‘ cbbL sequences Fig. 4: 1: soil with rye crop (Halle soil) without fertilization, 2: Halle soil with animal manure, 3: Halle soil with mineral fertilizer, 4: Sinorhizobium meliloti (positive control), M: MWM cbbL R sequences amplified from the different soil samples are distributed all over the red-like phylogenetic group discovery of yet undetected cbbL R sequences Fig. 3: Phylogenetic tree based on ‘green- like‘ cbbL sequences 2) 1)