Proteome analyses of hydrogen-producing hyperthermophilic archaeon Thermococcus onnurineus NA1 in different one-carbon substrate culture conditions

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.

Fig. 1.

A putative scheme of the cellular metabolism in the hydrogen producing arachaeon T. onnurineus NA1. A, glycolysis and gluconeogenesis. B, pyruvate degradation. C, pentose phosphate synthesis and carbon dioxide fixation. D, pseudo-TCA cycle. E, amino acid degradation and aldehyde metabolism. F, propionate utilization. Dashed lines or faint italic letters represent pathways or enzymes not yet experimentally validated in Thermococcales species. Each protein with its predicted function in ion or solute transport, or H₂ evolution or consumption, is illustrated on the membrane. Blue, red, and gray indicate proteins expressed prominently during growth on formate, CO, and starch, respectively. Major metabolic proteins deduced from the annotatable coding sequences (CDSs) were categorized by their induction on different substrates: formate (blue), CO (red), and starch (black). Other enzymes of the Embden-Meyerhof-Parnas pathway are designated by purple text, although they were mainly induced during growth on starch. Enzymes involved in sulfur reduction or ATP synthesis are shown in green, irrespective of their substrate specificity. Most proteins identified in T. onnurineus NA1 are shared by other Thermococcales: NPSOR (TON_0129), NADH:polysulfide oxidoreductase; FNR (TON_0056), ferredoxin-NADP(+) reductase; braC-4 (TON_0162), branched chain amino acid transporter; CbiOQ (TON_0243), ABC-type cobalt transport system; FepBCD (TON_0296), ABC-type Fe³⁺-siderophore transporter; FbpABC (TON_0983 and TON_0985), ABC-type Fe³⁺ transporter; Pst (TON_1464 and TON_1551), hypothetical phosphate transporter; NhaC (TON_1517), Na⁺/H⁺ antiporter; MnhB (TON_1577), multisubunit Na⁺/H⁺ antiporter; AppABC/OppBCDF (TON_1764 and TON_1768), ABC-type dipeptide/oligopeptide transporter; MalEFGK (TON_1791 and TON_1795), ABC-type maltodextrin transporter; AAT, additional amino acid ABC transporter; Fdred, reduced ferredoxin; Fdox, oxidized ferredoxin. The abbreviations of all of the identified proteins are presented separately in Table II and supplemental Table S1.

Fig. 2.

Expression map of hydrogenase gene cluster proteins. The expression map was constructed using the proteome of T. onnurineus NA1 cultivated with formate, CO, and starch as carbon and/or energy sources. Blue, red, and black arrows indicate proteins expressed prominently during growth on formate, CO, and starch, respectively. The white arrow indicates an unidentified gene product. The numbers below the colored arrows indicate the gene identification numbers of hydrogenase induced during growth.