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Investigation of Cell Envelope‐Associated Proteins in Rumen Methanogens as Targets for Understanding Host‐Microbe Interactions
Author(s) -
Yeung Juliana H.,
Shu Dairu,
Ferguson Timothy,
Altermann Eric,
Khanum Sofia,
Gupta Sandeep,
Loo Trevor S.,
Sutherland-Smith Andrew J.,
Heiser Axel,
Janssen Peter H.,
Wedlock D. Neil
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.08838
Subject(s) - methanogen , rumen , biology , archaea , biochemistry , bacteria , escherichia coli , recombinant dna , cell envelope , membrane protein , microbiology and biotechnology , gene , genetics , fermentation , membrane
The rumen microbiota is complex and almost totally composed of anaerobic organisms, including methanogenic archaea (methanogens). The latter occupy a unique niche in the rumen, using hydrogen, formate and methyl compounds to produce methane. Many of the proteins predicted from analysis of genome sequences of rumen‐dwelling methanogens, in particular cell envelope‐associated proteins, have no known biological function. To elucidate the role of these proteins in methanogen activity in the rumen and in the interaction of these archaea with other ruminal microbes, we have produced antibodies against selected methanogen proteins. The proteins were expressed in a standard bacterial expression system. An important first step was to determine if recombinantly produced proteins can generate antibodies that bind and recognise the native methanogens proteins. Bioinformatic analysis of the genome of Methanobrevibacter ruminantium M1 identified suitable cell surface‐located proteins for the studies. Predicted extracellular domains of these proteins were synthesised in Escherichia coli and circular dichroism analysis was performed to check that each recombinant protein had an ordered structure. Antibodies were produced against each recombinant protein and used in ELISA and Western blotting on lysates and membrane protein enriched fractions prepared from M1 cells. Proteins in the fractions were further separated by 2D gel electrophoresis and specific binding of the antibodies to the native targets was confirmed by LC‐MS/MS. Binding of the antibodies to methanogen cells was demonstrated using ELISA and flow cytometry. Our results to date suggest that expression of some of the cell surface proteins in bacteria produced recombinant proteins that sufficiently mimic the native proteins, with cross‐reactivity of antibodies to methanogen cell extracts. Our immunological studies, together with new information on gene expression from transcriptomic analysis of M1 in cultures, is being used to further understand the biological function of methanogen cell surface proteins and how methanogens interact with other microbes in the rumen. Support or Funding Information This research was funded jointly by Pastoral Greenhouse Gas Research Consortium (PGgRc) and New Zealand Agricultural Greenhouse Gas Research Centre (NZAGRC)