Deciphering metatranslatomes - the chain link to understand functionality of gut microbiota
Final Report Abstract
Bacterial gene regulation occurs at multiple levels. Genes encoded on DNA are translated into messenger RNA (transcription) and are translation into proteins in the ribosome (translation). Although it is now possible to determine the amount of protein (the proteome) in bacterial cultures, there are certain limitations as the chemistry of all amino acids is very different. Much simpler is sequencing of all RNAs of a cell (RNA-Seq). However, the amount of messenger RNA is only partially suitable for predicting the amount of protein, since the second level of regulation is omitted. The so-called Ribo-Seq, the measurement of "ribosome footprints", offers new possibilities: Here, after appropriate preparation, only the part of the messenger RNAs that is hidden in the ribosomes is sequenced. Bioinformatics is then used to determine which genes they come from. We were able to show that the amount of ribosome footprints correlates very well with the amount of protein. Ribo-Seq also allows discovering new gene-coding regions in the genome, especially those that produce small proteins. Due to the enrichment in the course of the experiment, weakly expressed genes can also be detected better. So far, Ribo-Seq has been carried out almost exclusively on individual bacteria, but in nature there are almost only microbial communities, such as in the intestine. Understanding gut bacteria and their function is a growing area of research, but functional assignments (which bacterium does what) are still in their infancy. The complexity of natural gut consortia is a major challenge for research. Minimal consortia with only a few bacterial species significantly reduce the complexity. Here we combined the use of minimal bacterial consortia with Ribo-Seq. The representation of the so-called meta-translatomes allows recording individual gene regulation at the level of the translatome of bacteria in communities. These meta-translatome measurements are superior to meta-proteomes because small and weak proteins (more precisely their translation) can also be measured. We were able to show that bacterial species, even when cultivated together, show a highly individual pattern of gene regulation, even in their central metabolism. Measurements from consortia originating from a host organism (here mouse) showed the regulation of genes that were only rarely observed in culture. The initially necessary methodical optimizations thus open up new research possibilities in minimal consortia, which will allow a better understanding of the interaction between intestinal bacteria and host.
Publications
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Metatranslatomes of murine microbiota. Kick-off Seminar CRC1371; 15.- 17.11.2017, Raitenhaslach, Germany.
Giehren F.
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Footprints in bacteria – Ribosome covered mRNAs pieces are better indicators for protein expression than RNAseq data. VAAM 2018, Wolfsburg, Germany.
Giehren F., Kreitmeier M., Fischer S., Adern Z. & Neuhaus K.
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RIBOseq and RNAseq experiments of anaerobic versus aerobic cultivation conditions of Escherichia coli LF 82; Campus Lab Seminar; 11.04.2018, TUM Weihenstephan, Freising, Germany.
Giehren F.
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Ribosome profiling and RNA-Seq of two gut bacteria in a mixed culture under aerobic and anaerobic growth-conditions. Quadram Institute Seminar; 16.09.2019, TUM Weihenstephan, Freising, Germany.
Giehren F.
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Ribosome profiling combined with RNA-Seq of Escherichia coli LF82 and Enterococcus faecalis OG1RF in a mixed culture under aerobic and anaerobic growth-conditions. RNA 2019 - 24th Annual Meeting of the RNA Society, Krakau, Poland.
Giehren F., Sewald Z., Mix L. & Neuhaus K.
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Ribosome profiling reveals novel small proteins in the gut bacterium Enterococcus faecalis OG1RF. Small Proteins in Prokaryotes, an Unexplored World; 16.-18.3.2020; Hamburg, Germany.
Giehren F., Sewald Z., Glaub A., Huptas C. & Neuhaus K.
