Revealing the insoluble metasecretome of lignocellulose-degrading microbial communities

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.

A robust, cost‐effective method for DNA, RNA and protein co‐extraction from soil, other complex microbiomes and pure cultures

“…As the novelty of our method lies in the recovery of proteins from the phenol phase, typically discarded in the protocol of Griffiths et al (), we further demonstrate that our biomolecule co‐extraction is suitable for metaproteomics analysis of other soil types with varying clay contents. Indeed, the DNA and RNA co‐extraction protocol from Griffiths et al () has been cited to date by over 1,250 papers including many reporting on downstream high‐throughput sequencing, including 16SrRNA and ITS amplicon analysis (Haas et al, ; Morawe et al, ; Sayer et al, ; Whitman et al, ), metagenomics (Malik, Thomson, Whiteley, Bailey, & Griffiths, ; Soares et al, ; Wilhelm, Hanson, Chandra, & Madsen, ) and metatranscriptomics (Alessi et al, ; de Menezes, Clipson, & Doyle, ; Hesse et al, ). Here, 16S rRNA profiling from DNA and cDNA was performed on three samples corresponding to three biological replicates from one soil type (with a clay content of 11.1%) while metaproteomics was conducted for nine samples corresponding to three biological replicates from three soil types (with clay content of 11.1%, 19.4% and 35.1%).…”

A microdialysis perspective of soil nitrogen availability

“…Recent functional studies of complex soil microbial communities are analysing metaexoproteomes, or metasecretomes, enabled by sensitive instrumentation and bioinformatics tools (Johnson-Rollings et al, 2014, Alessi et al, 2017. The increasing recognition that green plants are mixotrophs with an autotrophic shoot and a heterotrophic root (reviewed by Selosse et al, 2017 has also focused much attention on root exoenzymes and their roles in plant nutrition (reviewed by Paungfoo-Lonhienne et al, 2012).…”

“…Because of this, such enzyme techniques are widely recognised as providing only a 'potential' measure of activity under optimum incubation conditions (Burns et al, 2013). In situ approaches that can quantify enzyme activity in intact soils are highly sought after, and has led to the development of method such as zymography (Dong et al, 2007, Spohn et al, 2013, Spohn and Kuzyakov, 2014, Hofmann et al, 2016, as well as genetic / transcriptomic (Garoutte et al, 2016) and proteomic approaches (Schulze et al, 2004, Alessi et al, 2017. Although each method has benefits and weaknesses (discussed further in Chapter 5), new methods which can investigate the dynamics of enzyme production and activity at microscale environments are highly desirable, as are methods which can differentiate between distinct soil enzyme locations -those free in soil solution, and those which are bound to soil surfaces (Wallenstein and Weintraub, 2008).…”

“…Additionally, posttranslational modifications, re-assimilation and short life-spans of free enzymes may mean transcriptional information does not necessarily correlate with enzyme activity (Nannipieri et al, 2012, Rocca et al, 2014. However, ongoing advances are being made in understanding and linking transcription products with the metaexoproteome in complex matrices (Alessi et al, 2017). Proteomic approaches can directly identify a diversity of soil proteins including enzymes (Schulze et al, 2004, Alessi et al, 2017, but detection can be hampered by high turnover of proteins, along with low concentrations and heterogeneity in soil (Giagnoni et al, 2011, Nannipieri et al, 2012, Keiblinger et al, 2012.…”

“…However, ongoing advances are being made in understanding and linking transcription products with the metaexoproteome in complex matrices (Alessi et al, 2017). Proteomic approaches can directly identify a diversity of soil proteins including enzymes (Schulze et al, 2004, Alessi et al, 2017, but detection can be hampered by high turnover of proteins, along with low concentrations and heterogeneity in soil (Giagnoni et al, 2011, Nannipieri et al, 2012, Keiblinger et al, 2012. Dissolved phenolic compounds present in soils can also interfere with common protein quantification methods Jones, 2008, Redmile-Gordon et al, 2013).…”