Abstract
Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, is capable of H(2)-producing growth, considered to be hydrogenogenic carboxydotrophy. Utilization of formate as a sole energy source has been well studied in T. onnurineus NA1. However, whether formate can be used as its carbon source remains unknown. To obtain a global view of the metabolic characteristics of H(2)-producing growth, a quantitative proteome analysis of T. onnurineus NA1 grown on formate, CO, and starch was performed by combining one-dimensional SDS-PAGE with nano UPLC-MS(E). A total of 587 proteins corresponding to 29.7% of the encoding genes were identified, and the major metabolic pathways (especially energy metabolism) were characterized at the protein level. Expression of glycolytic enzymes was common but more highly induced in starch-grown cells. In contrast, enzymes involved in key steps of the gluconeogenesis and pentose phosphate pathways were strongly up-regulated in formate-grown cells, suggesting that formate could be utilized as a carbon source by T. onnurineus NA1. In accordance with the genomic analysis, comprehensive proteomic analysis also revealed a number of hydrogenase clusters apparently associated with formate metabolism. On the other hand, CODH and CO-induced hydrogenases belonging to the Hyg4-II cluster, as well as sulfhydrogenase-I and Mbx, were prominently expressed during CO culture. Our data suggest that CO can be utilized as a sole energy source for H(2) production via an electron transport mechanism and that CO(2) produced from catabolism or CO oxidation by CODH and CO-induced hydrogenases may subsequently be assimilated into the organic carbon. Overall, proteomic comparison of formate- and CO-grown cells with starch-grown cells revealed that a single carbon compound, such as formate and CO, can be utilized as an efficient substrate to provide cellular carbon and/or energy by T. onnurineus NA1.
Highlights
From the ‡Division of Life Science, Korea Basic Science Institute, Daejeon 305-806, Republic of Korea, §Gwangju Center, Korea Basic Science Institute, Gwangju 500-757, Republic of Korea, ¶Korea Ocean Research and Development Institute, Ansan 426-744, Republic of Korea, and the Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea
The maximal yield of H2 was achieved on formate or CO [22]. These results indicate that formate and CO can serve as major energy sources for T. onnurineus NA1
Identification of Proteins Expressed during Growth on Different Substrates—To obtain a more complete picture of the metabolism involved in growth, a genome-wide proteome analysis of T. onnurineus NA1 grown on formate, CO, and starch was performed by one-dimensional SDS-PAGE sample fractionation and LC-MSE-based protein identification
Summary
Strain and Culture Conditions—T. onnurineus NA1 [10] was cultured in yeast extract/peptone/sulfur medium that contained 35.0 g/liter NaCl, 3.3 g/liter Na2SO4, 0.05 g/liter KCl, 0.02 g/liter H3BO3, 8.8 g/liter MgCl21⁄76H2O, 0.05 g/liter CaCl21⁄72H2O, 0.61 g/liter N-(1,1dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, 0.001 g/liter resazurin, 3.0 g/liter yeast extract, 3.0 g/liter peptone, and 10.0 g/liter elemental sulfur. After drying in a speed vacuum concentrator (Mivac Quattro; Genevac Ltd.), the gels were incubated in a solution containing 10 mM DTT in 100 mM ABC at 56 °C for 1 h to reduce protein disulfide bonds and in the same volume of 55 mM iodoacetamide in 100 mM ABC added to alkylate cysteines in the dark for 45 min. The digested peptides were recovered by extracting twice with a solution containing 50 mM ammonium bicarbonate, 50% ACN, and 5% TFA. LC-MSE data were processed and searched using ProteinLynx GlobalServer version 2.3.3 (Waters Corporation). Analysis of quantitative changes in protein abundance, based on measurements of peptide ion peak intensities observed in low collision energy mode (MS) in a triplicate set, was carried out using ExpressionTM software (version 2). The Expression software generated results as an exact mass retention time table with quantitative protein and peptide values
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