Abstract
Microbial communities metabolize plant biomass using secreted enzymes; however, identifying extracellular proteins tightly bound to insoluble lignocellulose in these microbiomes presents a challenge, as the rigorous extraction required to elute these proteins also lyses the microbes associated with the plant biomass releasing intracellular proteins that contaminate the metasecretome. Here we describe a technique for targeting the extracellular proteome, which was used to compare the metasecretome and meta-surface-proteome of two lignocellulose-degrading communities grown on wheat straw and rice straw. A combination of mass spectrometry-based proteomics coupled with metatranscriptomics enabled the identification of a unique secretome pool from these lignocellulose-degrading communities. This method enabled us to efficiently discriminate the extracellular proteins from the intracellular proteins by improving detection of actively secreted and transmembrane proteins. In addition to the expected carbohydrate active enzymes, our new method reveals a large number of unknown proteins, supporting the notion that there are major gaps in our understanding of how microbial communities degrade lignocellulosic substrates.
Highlights
Understanding how plant biomass is degraded in soil and compost by mixed microbial communities, has been greatly advanced by the application of ‘omics’ technologies, in determining the way in which the metasecretome allows these communities to interact with one another and their surrounding environment[1,2,3,4,5,6]
We describe a methodology, which has allowed an unprecedented depth of analysis of the proteins present in the metaesecretomes of lignocellulose-degrading mixed microbial communities derived from wheat straw and rice straw compost, respectively
The composting communities are, dependent on the presence of a diverse range of actively secreted extracellular proteins involved in plant cell wall degradation and cell-associated transport proteins for rapid nutrient uptake[5, 24]
Summary
Understanding how plant biomass is degraded in soil and compost by mixed microbial communities, has been greatly advanced by the application of ‘omics’ technologies, in determining the way in which the metasecretome allows these communities to interact with one another and their surrounding environment[1,2,3,4,5,6]. Developing a robust method for metasecretome analysis of lignocellulose-degrading communities in environments such as soil or compost is challenging because many of the proteins involved in plant cell wall degradation are often tightly bound to the biomass[10]. To date, these bound proteins have been difficult to analyze because the stringent conditions needed to extract them generally leads to cell lysis and extensive contamination of the metasecretome with intracellular proteins. We report the development of a targeted methodology for metasecretome and meta-surface-proteome extraction and proteomic analysis of compost-derived mixed microbial consortia grown on wheat and rice straw This methodology, in combination with RNA-seq, led to identification of proteins putatively involved in lignocellulose degradation and nutrient transport from a diverse microbial community
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