Exploring the Molecular Machinery of Denitrification in Haloferax mediterranei Through Proteomics

Many proteins and enzymes involved in denitrification in haloarchaea can be inferred to be located between the cytoplasmic membrane and the S-layer, based on the presence of a Tat signal sequence and the orientation of the active site that some of these enzymes have. The membrane fraction of the hal...

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Bibliographic Details
Published in:Frontiers in microbiology 2020-12, Vol.11, p.605859-605859
Main Authors: Torregrosa-Crespo, Javier, Pire, Carmen, Richardson, David J, Martínez-Espinosa, Rosa María
Format: Article
Language:English
Subjects:
anaerobiosis
denitrification
detergents
haloarchaea
Haloferax
liquid chromatography-mass spectrometry
Microbiology
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Summary:Many proteins and enzymes involved in denitrification in haloarchaea can be inferred to be located between the cytoplasmic membrane and the S-layer, based on the presence of a Tat signal sequence and the orientation of the active site that some of these enzymes have. The membrane fraction of the haloarchaeon (R-4), grown under anaerobic conditions in the presence of nitrate, was solubilized to identify the respiratory proteins associated or anchored to it. Using Triton X-100, CHAPS, and n-Octyl-β-d-glucopyranoside at different concentrations we found the best conditions for isolating membrane proteins in micelles, in which enzymatic activity and stability were maintained. Then, they were subjected to purification using two chromatographic steps followed by the analysis of the eluents by NANO-ESI Chip-HPLC-MS/MS. The results showed that the four main enzymes of denitrification (nitrate, nitrite, nitric oxide, and nitrous oxide reductases) in were identified and they were co-purified thanks to the micelles made with Triton X-100 (20% w/v for membrane solubilisation and 0.2% w/v in the buffers used during purification). In addition, several accessory proteins involved in electron transfer processes during anaerobic respiration as well as proteins supporting ATP synthesis, redox balancing and oxygen sensing were detected. This is the first characterization of anaerobic membrane proteome of haloarchaea under denitrifying conditions using liquid chromatography-mass spectrometry. It provides new information for a better understanding of the anaerobic respiration in haloarchaea.
ISSN:1664-302X
1664-302X
DOI:10.3389/fmicb.2020.605859