Exploring the Molecular Machinery of Denitrification in Haloferax mediterranei Through Proteomics

Abstract

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 Haloferax mediterranei (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 H. mediterranei 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.

Assessment of Haloferax mediterranei Genome in Search of Copper-Molecular Machinery With Potential Applications for Bioremediation

“…Genes coding for cupredoxin-containing proteins (i.e., cuproproteins) found in H. mediterranei genome are here grouped into the following subgroups: denitrification proteins (nitrite and nitrous oxide reductases), cytochrome c and multicopper oxidases, and small blue copper proteins from the amicyanin/pseudoazurin and plastocyanin families. Denitrification is an anaerobic pathway achieved by the enzymes nitrate reductase (NarGH), nitrite reductase (NirK), nitric oxide reductase (qNor), and nitrous oxide reductase (NosZ), being NirK and NosZ copper-containing proteins ( Figure 2 , yellow; Torregrosa-Crespo et al, 2020 ).…”

“…The genome of H. mediterranei strain ATCC 33500 was used as a model in this study. This strain has a small-size chromosome of 2.9 Mbp (61.1% of GC content) and three megaplasmids, ranging from 132 to 505 Kbp (pHME132, pHME322, and pHME505; Torregrosa-Crespo et al, 2020 ). The H. mediterranei genome sequences of reference for this work are deposited in NCBI GenBank with the accession numbers CP0139139 (chromosome, complete genome), CP039142 (pHME132), CP039141 (pHME322), and CP039140 (pHME505).…”

“…Several recent studies state that this species could be a good candidate for the design of bioremediation-based technologies to treat wastewaters, particularly brines or osmosis concentrates showing high salt concentrations and significant concentrations of nitrate, nitrite and (per)chlorate, compounds that are toxic for most living beings ( Nájera-Fernández et al, 2012 ; Martínez-Espinosa et al, 2015 ; Torregrosa-Crespo et al, 2020 ). Besides, molecular machinery for the potential removal of cadmium has also been identified from many haloarchaeal species ( Vera-Bernal and Martínez-Espinosa, 2021 ).…”

In Silico Analysis of the Enzymes Involved in Haloarchaeal Denitrification

“…The potential application of Haloferax mediterranei as a bioremediating agent in contaminated saline waters has been studied [21]. Haloferax mediterranei gas emission under denitrifying conditions has been measured, confirming that it is a complete denitrifier able to transform nitrate to dinitrogen even more efficiently than other Haloferax species [7,22]. Haloferax mediterranei NarGH and NirK have been studied at the biochemical level reporting expression in anaerobic conditions [20,21].…”

Distribution of Denitrification among Haloarchaea: A Comprehensive Study

“…In terms of respiration, due to lower oxygen solubility in these environments, some microorganisms have developed different strategies. Some examples are the production of gas vesicles (also called gas vacuoles) that enable cells to float to the surface of the brines where oxygen concentration is higher or the use of N-compounds as final electron acceptor, such as nitrate or nitrite using denitrification pathway [ 3 , 4 ].…”