Supporting Information

Lim et al. 10.1073/pnas.1407056111 SI Materials and Methods the reduction of 2 mmol methyl viologen/min, which is equivalent Composition of MM1 Medium. The MM1 medium had the following to 1 mmol of formate. composition (in grams per liter): extract (1), sodium formate Immunoblotting Assay. Twenty micrograms of the constructed (10), NaCl (35), KCl (0.7), MgSO4 (3.9), CaCl2·2H2O (0.4), inverted membrane vesicles were loaded and separated on 12% NH4Cl (0.3), Na2HPO4 (0.15), NaSiO3 (0.03), NaHCO3 (0.5), − cysteine·HCl (0.5), and resazurin (0.001); 1 mL·L 1 of Holden’s SDS gel. The gels were then transferred to nitrocellulose mem- − trace elements/Fe-EDTA solution (1) and 1 mL·L 1 of Balch’s brane using Trans-Blot Turbo Transfer System and Transfer Pack (Bio-Rad). Formate dehydrogenase (Fdh2), hydrogenase vitamin solution (2) were supplemented into the medium. The + + pH of the medium was adjusted to 6.5 with 2 N HCl. (Mfh2), and Na /H antiporter (Mnh2) in the inverted vesicles were detected by Western blot analysis using antisera toward Formate Dehydrogenase Assay. TON_1563 (formate dehydrogenase large subunit), TON_1569 Formate dehydrogenase activity + + was measured by a colorimetric assay with methyl viologen (MV) ([NiFe]-hydrogenase large subunit) and TON_1579 (Na /H −1 −1 as electron acceptor (e578 = 9.7 mM ·cm at 578 nm) (3) and antiporter subunit), respectively. TON_1563, TON_1569 and formate as electron donor. The assay was conducted in 1 mL TON_1579 were overexpressed and the proteins purified in volume of AA-buffer [10 mM bis(tris[hydroxymethyl]amino- . One milligram of each recombinant protein was methane) sulfate, 140 mM choline chloride, 5 mM MgSO4, and used to get antisera, performed by the standard protocol of 2 mM DTT, pH 6.5] containing 5 mM MV and 0.5 μg of inverted Young In Frontier Co. Western blots were prepared and ana- membrane vesicle in a serum-stoppered glass cuvette. The re- lyzed using a chemiluminescent dye with the Immun-Star HRP action mixture was preincubated at 60 °C for 10 min and then the chemiluminescent kit (Bio-Rad), and a quantitative imaging reaction was started by the addition of 50 mM sodium formate. system, Chemi Doc MP Imaging System, was used for detection One unit of activity is defined as the amount of catalyzing and analysis of chemiluminescence images.

1. Holden JF, et al. (2001) Diversity among three novel groups of hyperthermophilic deep- 3. Schmitz RP, Diekert G (2003) Purification and properties of the formate dehydrogenase sea Thermococcus species from three sites in the northeastern Pacific Ocean. FEMS and characterization of the fdhA gene of Sulfurospirillum multivorans. Arch Microbiol Microbiol Ecol 36(1):51–60. 180(6):394–401. 2. Balch WE, Wolfe RS (1976) New approach to the cultivation of methanogenic : 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressureized atmosphere. Appl Environ Microbiol 32(6):781–791.

Lim et al. www.pnas.org/cgi/content/short/1407056111 1of3 Fig. S1. Effect of ionophores and inhibitors on (A) Formate oxidation using 10 mM sodium formate, (B)H2 production using 150 mM potassium formate, and (C) ATP content. CuCl2 was used as a hydrogenase inhibitor, CCCP (carbonyl cyanide 3-chlorophenylhydrazone) and TCS (3,3′,4′,5-tetrachlorosalicylanilide) as + + protonophores, EIPA [5-(N-ethyl-N-isopropyl) amiloride] as Na /H antiporter inhibitor, and DES [(E)-3,4-bis(4-hydroxyphenyl)-3-hexene)] and DCCD (N,N′- − − dicyclohexylcarbodiimide) as ATP synthase inhibitor. Controls received the solvent only. One hundred percent corresponds to 29.4 U·mg 1·min 1 in A, 11.6 − − − mmol·L 1·h 1 in B, and 163 nmol·L 1 in C. The ATP content was determined 30 min after addition of 50 mM potassium formate.

Lim et al. www.pnas.org/cgi/content/short/1407056111 2of3 − Fig. S2. Growth of wild-type and mutant strains on ASW-YT (4 g·L 1 elemental sulfur) medium. Symbols indicate wild-type T. onnurinues NA1 (●), Δfdh2 (○), Δmnh2-1 (▼), Δmnh2-2 (△), and Δmnh2-3 (■).

Fig. S3. Determination of Fdh2, Mfh2, and Mnh2 in inverted membrane vesicles from T. onnurineus NA1 by immunoblotting analyses. Twenty micrograms of membrane vesicles were used for SDS/PAGE.

Lim et al. www.pnas.org/cgi/content/short/1407056111 3of3