In my PhD thesis I investigated two groups of microorganisms from soil habitats in an environmental genomic approach. Molecular phylogenetic studies based on the characterisation of 16S rRNA genes have shown that soil habitats are particularly rich in microbial diversity. In contrast, due to the lack of adequate techniques, more than 99% of the microorganisms are still uncharacterised, because they can not be cultivated in the laboratory. Therefore I have applied an environmental genomic approach to characterisemicroorganisms on their genomic level without the need of cultivation. My studies involved members of the archaeal kingdom crenarchaeota which were considered until recently as extremophiles, exclusively residing in hot environments and Acidobacteria, a novel phylum within the Bacteria, which shows a similar phylogenetic depth as that of the Proteobacteria. The development of a method based on a 2-phase gel-electrophoresis allowed the isolation of highly pure and cloneable high molecular weight DNA from soil by elimination of polyphenolic compounds (humic and fulvic acids) which heavily contaminate soil DNA (Quaiser et al., 2002a and 2002b). With these nucleic acids complex large insert libraries (metagenomic libraries) containing more than 4 Gbp of soil DNA from three different habitats were constructed. Characterisation of the metagenomic libraries revealed that a large microbial diversity predicted by 16S rDNA surveys was reflected on the genomic level. With Archaea-specific and Acidobacteria-specific probes one genomic fragment of a mesophilic uncultured Crenarchaeota and six genomic fragments of the Acidobacteria (210.000 bp) from two fosmid libraries were identified and characterised (Quaiser et al., 2002a; Quaiser et al., 2003). The archaeal clone contained a 34 kb fragment with a complete rRNA operon and 17 genes encoding putative proteins. Phylogenetic analysis of the rRNA genes and of a protein gene confirmed the affiliation of the genomic fragment to the group of non-thermophilic Crenarchaeota. The genome structure and gene content showed significant differences to its thermophilic cultivated relatives. Sequence analysis of the six acidobacterial genomic fragments resulted in more than 210 kbp of genomic information from this phylum (Quaiser et al., 2003). The 16S rRNA genes of the genomic fragments differed between 2.3 and 19.9% and were phylogenetically placed into two different subgroups of Acidobacteria (groups III and V). Although partial co-linearity was found between the genomic fragments, the gene content around the rRNA operons was generally not conserved. Phylogenetic reconstructions with orthologs that were encoded on two of the six genomic fragments confirmed the coherence of the acidobacterial phylum. One genomic fragment harboured a cluster of 8 genes which was syntenic and highly homologous to genomic regions in Rhodopseudomonas palustris and Bradyrhizobium japonicum, including a conserved two-component system. Phylogenetic analysis confirmed that this similarity between Rhizobiales and Acidobacteria might be due to a horizontal gene transfer. In total, these data gave first insights into the genome content and diversity of the ubiquitously distributed but poorly characterised mesophilic Crenarchaeota from soil and the phylum of Acidobacteria. Furthermore, novel techniques were established that allow to characterise and exploit uncultivated microorganisms from soil and sediment samples by genomic analyses and heterologous expression studies.