Final Activity Report Summary - ISOTONIC (Isotope tools to investigate structure and function of microbial communities)
The central aim of ISOTONIC was to combine the strength of cutting edge techniques in stable isotope tracer chemistry with modern genomic and proteomic analysis to improve the stable isotope probing (SIP) concept for the investigation of structure, function and activity of microbial communities. Stable isotope probing techniques are based on the fact that carbon and nitrogen of a substrate is used in biosynthesis for the production of biomass. Thus, stable isotope labelled substrates are transformed into stable isotope labelled biomass upon anabolism; the isotope label in different biomolecules (e.g. proteins, lipids, nucleic acids) can be used as an indicator for substrate metabolisation. In tracer experiment with isotope enriched substrates, lipids, nucleic acids and proteins will become labelled by stable isotopes upon biosynthesis. Therefore, isotope tracer techniques are excellent tools for process analysis.
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.
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.