The rumen microbial metaproteome as revealed by SDS-PAGE

Timothy J Snelling,R John Wallace

doi:10.1186/s12866-016-0917-y

Abstract

BackgroundRuminal digestion is carried out by large numbers of bacteria, archaea, protozoa and fungi. Understanding the microbiota is important because ruminal fermentation dictates the efficiency of feed utilisation by the animal and is also responsible for major emissions of the greenhouse gas, methane. Recent metagenomic and metatranscriptomic studies have helped to elucidate many features of the composition and activity of the microbiota. The metaproteome provides complementary information to these other –omics technologies. The aim of this study was to explore the metaproteome of bovine and ovine ruminal digesta using 2D SDS-PAGE.ResultsDigesta samples were taken via ruminal fistulae and by gastric intubation, or at slaughter, and stored in glycerol at −80 °C. A protein extraction protocol was developed to maximise yield and representativeness of the protein content. The proteome of ruminal digesta taken from dairy cows fed a high concentrate diet was dominated by a few very highly expressed proteins, which were identified by LC-MS/MS to be structural proteins, such as actin and α- and β-tubulins, derived from ciliate protozoa. Removal of protozoa from digesta before extraction of proteins revealed the prokaryotic metaproteome, which was dominated by enzymes involved in glycolysis, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, phosphoglycerate kinase and triosephosphate isomerase. The enzymes were predominantly from the Firmicutes and Bacteroidetes phyla. Enzymes from methanogenic archaea were also abundant, consistent with the importance of methane formation in the rumen. Gels from samples from dairy cows fed a high proportion of grass silage were consistently obscured by co-staining of humic compounds. Samples from beef cattle and fattening lambs receiving a predominantly concentrate diet produced clearer gels, but the pattern of spots was inconsistent between samples, making comparisons difficult.ConclusionThis work demonstrated for the first time that 2D-PAGE reveals key structural proteins and enzymes in the rumen microbial community, despite its high complexity, and that taxonomic information can be deduced from the analysis. However, technical issues associated with feed material contamination, which affects the reproducibility of electrophoresis of different samples, limits its value.

Highlights

Ruminal digestion is carried out by large numbers of bacteria, archaea, protozoa and fungi
Effects of different sample types on electrophoresis The ruminal metaproteome from a variety of species and sample types was characterised by SDS-PAGE using digesta samples obtained from a variety of ruminant species, including cattle, sheep and reindeer, fed different diets
The samples from the forage/barley concentrate fed dairy and beef cattle taken by gastric tube contained very little in the way of plant fibre and were processed as received

Summary

Introduction

Ruminal digestion is carried out by large numbers of bacteria, archaea, protozoa and fungi. Understanding the microbiota is important because ruminal fermentation dictates the efficiency of feed utilisation by the animal and is responsible for major emissions of the greenhouse gas, methane. The rumen is the primary digestive organ in ruminants such as cattle, sheep, buffaloes and deer. It contains a vast number of anaerobic eukaryotic and prokaryotic microorganisms, which break down ingested feed materials to short chain fatty acids that are absorbed, to be used by the host animal for energy [1, 2]. Understanding the composition and activity of the rumen microbial community is crucial if we are to improve productivity and to lessen the environmental impact associated with ruminant livestock. The rumen has a major influence on the health of the animal, so understanding the composition and function

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