The rumen microbial metaproteome as revealed by SDS-PAGE

Timothy J. Snelling, R. John Wallace

Research output: Contribution to journalArticle

10 Citations (Scopus)
5 Downloads (Pure)

Abstract

Background
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 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.

Results
Digesta 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.

Conclusion
This 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.
Original languageEnglish
Article number9
JournalBioMed Central Microbiology
Volume17
DOIs
Publication statusPublished - 7 Jan 2017

Fingerprint

Rumen
Polyacrylamide Gel Electrophoresis
Microbiota
Archaea
Methane
Electrophoresis, Gel, Two-Dimensional
Enzymes
Proteins
Gels
Gastric Fistula
Triose-Phosphate Isomerase
Bacteroidetes
Phosphoglycerate Kinase
Diet
Metagenomics
Phosphoenolpyruvate
Glyceraldehyde-3-Phosphate Dehydrogenases
Silage
Glycolysis
Proteome

Keywords

  • cattle
  • proteomics
  • rumen
  • sheep

Cite this

The rumen microbial metaproteome as revealed by SDS-PAGE. / Snelling, Timothy J.; Wallace, R. John.

In: BioMed Central Microbiology, Vol. 17, 9, 07.01.2017.

Research output: Contribution to journalArticle

@article{42fb40086d6241da896c2b11a467abf4,
title = "The rumen microbial metaproteome as revealed by SDS-PAGE",
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.",
keywords = "cattle, proteomics, rumen , sheep",
author = "Snelling, {Timothy J.} and Wallace, {R. John}",
note = "This work was supported by the RuminOmics project and funded by the European Commission (Grant Agreement No. 289319). The Rowett Institute is funded by the Rural and Environment Science and Analytical Services Division (RESAS) of the Scottish Government. The funding bodies had no role in the design of the study or collection, analysis, or interpretation of data or in writing the manuscript.",
year = "2017",
month = "1",
day = "7",
doi = "10.1186/s12866-016-0917-y",
language = "English",
volume = "17",
journal = "BioMed Central Microbiology",
issn = "1471-2180",
publisher = "BioMed Central",

}

TY - JOUR

T1 - The rumen microbial metaproteome as revealed by SDS-PAGE

AU - Snelling, Timothy J.

AU - Wallace, R. John

N1 - This work was supported by the RuminOmics project and funded by the European Commission (Grant Agreement No. 289319). The Rowett Institute is funded by the Rural and Environment Science and Analytical Services Division (RESAS) of the Scottish Government. The funding bodies had no role in the design of the study or collection, analysis, or interpretation of data or in writing the manuscript.

PY - 2017/1/7

Y1 - 2017/1/7

N2 - 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.

AB - 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.

KW - cattle

KW - proteomics

KW - rumen

KW - sheep

U2 - 10.1186/s12866-016-0917-y

DO - 10.1186/s12866-016-0917-y

M3 - Article

VL - 17

JO - BioMed Central Microbiology

JF - BioMed Central Microbiology

SN - 1471-2180

M1 - 9

ER -