Institute of Soil Ecology

1. b M 1 2 3 4 M 800 bp • low variability of the RFLP patterns • low diversity of cbbLG sequences • no detectable variation in pattern composition within differently managed soils HaSMRED143 HaSMRED8 HaSMRED75 manganese-oxidizing bacterium Nitrobacter winogradskyi IFO HaKORED8 Bradyrhizobium japonicum Nitrobacter winogradskyi AF HaSMRED139 UGREEN42 HaSMRED20 UGREEN7 HaKORED78 HaKORED36D Nitrobacter vulgaris Nitrosospira sp. AF UGREEN22 Nitrosospira sp. 40KI Nitrosospira sp. III2 UGREEN6 Nitrosospira sp. O13 UGREEN15 Nitrosospira sp. A4 UGREEN10 HaKORED32D HaNPKRED1 UGREEN13 HaSMRED2 UGREEN46 HaNPKRED7 UGREEN3 HaKORED15 HaKORED2 UGREEN11 HaSMRED1 UGREEN41 HaSMRED14 HaSMRED29 HGREEN2 HaKORED7 UGREEN9 HaKORED13, Nitrosospira sp. TCH716 HaSMRED46 HaKORED6 Halothiobacillus sp. RA13 HaSMRED12 Nitrosomonas sp. ENI-11 HaNPKRED16 Thiobacillus intermedius K12 Rhodobacter azotoformans Rhodobacter sphaeroides uncultured bacterium Sinorhizobium meliloti Acidithiobacillus ferrooxidans HaKORED22 gamma proteobacterium MLHE-1 HaKORED21 HaNPKRED5 Methylococcus capsulatus HaNPKRED20 uncultured bacterium HaKORED49b uncultured bacterium HaKORED5 HaKORED11 Hydrogenophaga pseudoflava HaSMRED4 Nitrosomonas europaea Ralstonia eutropha megaplasmid pHG1 Thiobacillus denitrificans Ralstonia eutropha HaNPKRED17 Thiobacillus sp. GREEN Ralstonia eutropha H850 uncultured bacterium Hawaii Lo1 Xanthobacter flavus uncultured bacterium Hawaii Lo2 0.10 Hydrogenovibrio marinus 1 Rhodobacter capsulatus Hydrogenophilus thermoluteolus endosymbiont of Pogonophora sp. Nitrosococcus halophilus Synechococcus sp. WH 7805 Synechococcus sp. Synechococcus sp. WH 8103 Synechococcus sp. WH 8101 uncultured Synechococcus sp. Synechococcus sp. WH 8103 uncultured Synechococcus sp. uncultured Synechococcus sp. uncultured Synechococcus sp. uncultured Prochlorococcus sp. Prochlorococcus marinus subsp. uncultured prochlorophyte 4DCH uncultured Prochlorococcus sp. Chloroplast uncultured cyanoba Hydrogenovibrio marinus Allochromatium vinosum RED Thermosynechococcus elongatus Synechococcus sp. Anabaena sp. Prochlorothrix hollandica Synechococcus PCC7002 0.10 Diversity of cbbL genes from autotrophic bacteria in differently managed agricultural soils Institute of Soil Ecology 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 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 CO2-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 CO2-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. Fig.1: cbbL based phylogenetic tree reflecting the affiliation of ‚green-like‘ and ‚red-like‘ sequences (modified from Watson et al., 1997) Results and Discussion Efficiency of cbbLGamplificationfrom soil Efficiency of cbbLRamplificationfrom soil M 1 2 3 4 M 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 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 1100 bp • successful detection of red-like cbbL fragments from soil • no differences in cbbL amplification depending on the kind of fertilization were observed • successful detection of green-like cbbL fragments from soil • differences in cbbL amplification depending on the kind of fertilization were observed RFLP analysis of cbbLG amplificates RFLP analysis of cbbLR amplificates • high variability of the RFLP patterns • low diversity of cbbLRsequencesin halle soil without ferilization (1), high diversity inHalle soilstreated with animal manure and mineral fertilizer (2) • significant variations in the composition of the cbbL-containing community in differently managed Halle soils 1) 2) Phylogenetic analysis of cbbLR Phylogenetic analysis of cbbLG • cbbLG sequences amplified from the different soil samples are closely related to cbbLG of Nitrobacter vulgaris and N. winogradskyi • cbbLR sequences amplified from the different soil samples are distributed all over the red-like phylogenetic group • discovery of yet undetected cbbLR sequences Fig. 5: Phylogenetic tree based on ‘red-like‘ cbbLsequences Fig. 3: Phylogenetic tree based on ‘green-like‘ cbbLsequences References: Kusian, B. and Bowien, B. 1997 Organization and regulation of cbb CO2 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. • Perspectives • Quantification of ‘green-like’ and ‘red- like’ cbbL genes by TaqMan-PCR • Assessment of the cbbL transcript levels • Effect of H2-treatment on the diversity of cbbL genes Acknowledgement: This study is supported by the Deutsche Forschungsgemeinschaft (SPP1090, Ha 1708/6).