Final Activity Report Summary - ISOTONIC (Isotope tools to investigate structure and function of microbial communities)
We applied modern molecular biological techniques to analyse the composition of environmental microbial communities in order to identify distinct members of these communities. For unravelling their function and activity we developed the technique of protein stable isotope probing (Protein SIP). Proteins are the biochemical catalysts of microbial processes, reflecting the in situ activity of microorganisms. The isotope label in lipids was found to be a very sensitive tracer for metabolic activity; however, the taxonomic and functional value of lipids is limited and did not allow analysing structure and function of microbial communities. The isotope label in proteins or nucleic acids contains important information on function and activity of the host organism.
In the course of this project we developed concepts to exploit the information of stable isotope probing of proteins and DNA/RNA to investigate the structure, function and activity of microbial communities and to elucidate tropic levels in food webs. We developed a stable-isotope probing concept in order to link microbe-specific metabolic functions with phylogenetic information. Carbon or nitrogen labelled substrates (typically with >98% heavy label) are commonly used in cultivation experiments and the heavy isotope incorporation into proteins is analysed employing modern techniques in protein mass spectrometry.
The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. Our results show that stable isotope probing of nucleic acids and proteins is, due to improved separation techniques, a powerful toolbox for linking microbial community structures with in situ functions of individual community members.
The technique allows investigating degradation processes and food webs in microbial communities. The proteomic analysis of labelled proteins allows identifying proteins and characterising their molecular properties and provides clues to elucidate their function within host organisms in microbial biofilms.