Abstract
The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been shown to produce H2 when using CO, formate, or starch as a growth substrate. This strain can also utilize elemental sulfur as a terminal electron acceptor for heterotrophic growth. To gain insight into sulfur metabolism, the proteome of T. onnurineus NA1 cells grown under sulfur culture conditions was quantified and compared with those grown under H2-evolving substrate culture conditions. Using label-free nano-UPLC-MSE-based comparative proteomic analysis, approximately 38.4% of the total identified proteome (589 proteins) was found to be significantly up-regulated (≥1.5-fold) under sulfur culture conditions. Many of these proteins were functionally associated with carbon fixation, Fe–S cluster biogenesis, ATP synthesis, sulfur reduction, protein glycosylation, protein translocation, and formate oxidation. Based on the abundances of the identified proteins in this and other genomic studies, the pathways associated with reductive sulfur metabolism, H2-metabolism, and oxidative stress defense were proposed. The results also revealed markedly lower expression levels of enzymes involved in the sulfur assimilation pathway, as well as cysteine desulfurase, under sulfur culture condition. The present results provide the first global atlas of proteome changes triggered by sulfur, and may facilitate an understanding of how hyperthermophilic archaea adapt to sulfur-rich, extreme environments.
Highlights
Thermococcales are strictly anaerobic and heterotrophic hyperthermophiles that can utilize various complex substrates such as yeast extract, peptone, and amino acids such as carbon sources, and grow optimally at temperatures between 80 and 100 °C, depending on the species
These results indicate that T. onnurineus NA1 cells undergo a shift in cell metabolism for utilization of S° as an electron acceptor, switching the production of H2 to H2S
It was expected that comparative analysis of the proteomic responses to sulfur and the three growth substrates (CO, formate, and starch) would enable the identification of a set of candidate proteins involved in sulfur metabolism, as well as the uncovering of a metabolic switch between sulfur reduction and H2 production in hyperthermophilic archaea
Summary
Thermococcales are strictly anaerobic and heterotrophic hyperthermophiles that can utilize various complex substrates such as yeast extract, peptone, and amino acids such as carbon sources, and grow optimally at temperatures between 80 and 100 °C, depending on the species. Owing to the discovery of new enzymes, several distinct hydrogenase gene clusters, and their regulatory ability, it shows great potential for use in biotechnology [17,18,19] This metabolic versatility, together with the significant potential for biotechnology uses, would provide a good platform to study sulfur-dependent metabolic changes, as well as to examine the activities of hydrogenases, sulfur reductase, and electron transfer occurring in T. onnurineus NA1, at both the protein and genetic levels. SDS-PAGE/LC-MS/MS shotgun proteomic analysis revealed that metabolic enzymes specific for hydrogen production were prominently expressed when T. onnurineus NA1 cells were cultured under carboxydotrophic conditions rather than under organotrophic conditions (yeast extract-peptone-sulfur) [29]. This report gives basic insight into the metabolic adaptation of hyperthermophilic archaea in response to sulfur at the protein level, and provides a sound basis for further physiological and biochemical studies
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