Browsing by Person "Deusch, Simon"

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Metaomic studies of the dietary impact on the structural and functional diversity of the rumen microbiome
(2018) Deusch, Simon; Seifert, Jana
Ruminant production efficiency and related emission of greenhouse gases are mainly determined by the rumen microbiome. The structure and activity of the microbial communities in turn are mostly influenced by the animal’s feed intake. The most widely used forage sources for ruminant production in Europe are corn silage, grass silage and grass hay. Progress in animal production requires optimized feeding strategies which presuppose an improved understanding of the dietary impact on the complex bionetwork residing in the rumen. A broad range of different methods are applicable to investigate archaea and bacteria which represent the most active members of the rumen microbiome. Most rumen studies available are restricted to nucleic acid-based approaches with limited functional insights. To improve knowledge about the prokaryotic communities and their adaptation responses to different animal feeds, it is essential to focus on the actual functions out of numerous possibilities that are encoded by the genomes of the rumen microbiome. Therefore proteins are best suited since representing the actual function of investigated cells combined with phylogenetic information. The major aim of this project was the feasible, first-time establishment of a metaproteomics-based characterization of the ruminal prokaryotic communities to further investigate the dietary impact on the prokaryotic rumen metaproteome. The first part was providing an overview about research that used state of the art technologies to investigate the microbiome of the gastrointestinal tract of farm animals. Yet, Omics-technologies and their combination are rarely employed in livestock science. The considered studies relied mainly on stand-alone, DNA-based molecular methods which clearly emphasized the importance of introducing contemporary methods such as shotgun metaproteomics to study the rumen microbiome and to gain deeper, more complete insights into the actual functions carried out by the specific members of the prokaryotic communities. The second part of the current project focused on a suitable, mass spectrometry-based analysis of the prokaryotic communities in the rumen ecosystem. Metaproteomic studies are challenged by the heterogeneity of the rumen sample matter that contains, besides archaeal and bacterial cells, also eukaryotic cells of rumen fungi and protozoa as well as enormous amounts of plant cells from ingested feed and epithelial cells of the animals. Shotgun metaproteomic studies require the extraction of proteins preferably of the desired target organisms to increase the coverage of the respective metaproteome and the reliability of subsequent protein identifications. This entails the avoidance of undesired proteins present in the rumen samples. In contrast to nucleic acids, proteins cannot be enriched or amplified by PCR thus, optimized sample preparation protocols are necessary in order to retrieve enhanced amounts of prokaryotic instead of plant-derived or other eukaryotic cells before protein extraction and subsequent LC-MS/MS analysis. The final step and the major aim of this project was the in depth analysis of the metaproteome of archaea and bacteria and their adaptive response to the most common forages, corn silage, grass silage and grass hay accessing as well host-related influences and variations between different ecological niches within the rumen. Improved mass-spectrometric measurements and the construction of a customized, sample-specific in-house database for enhanced bioinformatic quantification of proteins yielded comprehensive datasets comprising 8,163 bacterial and 358 archaeal proteins that were identified across 27 samples from three different rumen fractions of three Jersey cows, fed rotationally with three different diets. The functional and structural data of the metaproteomic analysis was further flanked by 16S rRNA gene-based analyses of the archaeal and bacterial community structures and the metabolomes of the rumen fluid fractions were quantified by nuclear magnetic resonance. So far, to the best of our knowledge, there are no studies investigating the metaproteome expressed by the entirety of archaeal and bacterial communities in the different phases of the rumen ecosystem under varying dietary influence. Dietary treatments revealed significant variations in the metaproteome composition and community structures of ruminal bacteria. Host-related effects were not significant. In conclusion, within this project the application of shotgun metaproteomics to characterize the prokaryotic rumen metaproteome was successfully implemented and the obtained results clearly emphasized the benefits of using complementary, state of the art methods to study the microbiome of complex ecosystems like the rumen. Considering the specific functional niches of the rumen microbiome have been shown to be of great importance.