Abstract
BackgroundIn silico, secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences. For metasecretome (collection of surface, secreted and transmembrane proteins from environmental microbial communities) this approach is impractical, considering that the metasecretome open reading frames (ORFs) comprise only 10% to 30% of total metagenome, and are poorly represented in the dataset due to overall low coverage of metagenomic gene pool, even in large-scale projects.ResultsBy combining secretome-selective phage display and next-generation sequencing, we focused the sequence analysis of complex rumen microbial community on the metasecretome component of the metagenome. This approach achieved high enrichment (29 fold) of secreted fibrolytic enzymes from the plant-adherent microbial community of the bovine rumen. In particular, we identified hundreds of heretofore rare modules belonging to cellulosomes, cell-surface complexes specialised for recognition and degradation of the plant fibre.ConclusionsAs a method, metasecretome phage display combined with next-generation sequencing has a power to sample the diversity of low-abundance surface and secreted proteins that would otherwise require exceptionally large metagenomic sequencing projects. As a resource, metasecretome display library backed by the dataset obtained by next-generation sequencing is ready for i) affinity selection by standard phage display methodology and ii) easy purification of displayed proteins as part of the virion for individual functional analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-356) contains supplementary material, which is available to authorized users.
Highlights
In silico, secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences
To maximise the probability of identifying extracellular proteins involved in fibre degradation, a plant-adherent fraction of the rumen microbial community from pasture-fed cows was used as a source of DNA for library construction
The metasecretome phage display method combined with next-generation sequencing has the power to functionally select for, and reveal, the diversity of low-abundance surface and secreted proteins that would otherwise require large metagenomic sequencing efforts to reveal
Summary
Secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences. For metasecretome (collection of surface, secreted and transmembrane proteins from environmental microbial communities) this approach is impractical, considering that the metasecretome open reading frames (ORFs) comprise only 10% to 30% of total metagenome, and are poorly represented in the dataset due to overall low coverage of metagenomic gene pool, even in large-scale projects. Microorganisms account for a major proportion of our planet’s biological diversity and present an enormous and largely unknown resource that can be utilised in the discovery of novel genes, bioactive molecules [1] and new biocatalysts. These may be exploited to improve industrially relevant processes [2]. It is estimated that the rumen harbours up to 3,000 bacterial species, the majority belonging to the phyla Firmicutes and Bacteroidetes, with species belonging to the Proteobacteria, Fibrobacteres and Spirochaetes present [12,13,14,15]
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