GGAAATCCCTTAAAAGTCG TCTCA GAGCTGCGCTCGCCC GTGCCTGAAAACCGAAGA GG ACCAACGTGTCAGTGCTTT TTG GCATCCACACCGCATGG

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Edward Manning
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1 Table S1. Sequences of primers described in this report. Gene (loci number, predicted/ reported function) Forward primer (5 3 ) Reverse primer (5 3 ) 16S rrna gene GGAAATTTAAAAGTG TTA GATTATAGGATTAA TTA glta (T1834, citrate synthase) AAGAATTGTGTG acla (T88, AL α-subunit) TTAAAAGTGGGG aclb (T89, AL β-subunit) GGTTTGTAG GTGGTGGGTGA AATTTGGGGAA GGAGGAATTTAGA pora (T1628, pyruvate: Fd oxidoreductase, PFOR) korb (T162, KFOR, β subunit) kora (T163, KFOR, α subunit ) GAGTGGTG GTGTGAAAAGAAGA GG AAAGTGTAGTGTTT TTG ATTTTGAGAGTTAA GA GGAGTTGAGAGAG GGGTGGGTAAGTG acn (T543, aconitase) AAGGAGTTA AAAATAGGGAGGTTGG T icd (T351, isocitrate dehydrogenase) ppd (T1682, pyruvate phosphate dikinase) GATAAGATGG GATATAGAAAATTA TGA GGTATATAGAAAGTG G ATAGTTGATTTTTTTA GG ppc (T164, PEP carboxylase) GAGAATAAGGTA GTA TTTTGGAGAGAATTTG pcka (T2232, PEP carboxykinase) acsa (T1652, acetyl-oa synthase) GGTGATATTAAAT GGT GAAAGTGATTTG AATTTGGGGATGG TGAGTGTTGATTTGG acka (T1525, acetate kinase) TGGAGTAAA TTTGGGATGGAGTAAG pta (T85, phosphotransacetylase) sdhb-like (T41, succinate/ fumarate oxidoreductase, subunit B) sdha-like (T42, succinate/ fumarate oxidoreductase, subunit A) GTGGTGAATATTTG A TGAGAAAAAGGGAAG TG ATGTGGTGGTGGG TGAGGGTTGGG GGATGATTGGTTG GGATTGGTTTATTGT 1

2 sdhb (T2266, succinate dehydrogenase, subunit B) sdha (T2267, succinate dehydrogenase, subunit A) AAGTAATG AGGAAAAAAGG GGGAAGAGAGGAGGTG AAGAGAAG 2

3 Figure S1. Mass spec of Bhl c obtained from cultures grown on unlabeled acetate or pyruvate (A), [1-13 ] acetate (B), [2-13 ]acetate (), [1-13 ]pyruvate (D) and [3-13 ]pyruvate (E). The 13 -enriched Bhl c was detected with labeled acetate or pyruvate, as shown in panels (B) to (E). A E B E

4 E D E

5 E E

6 Figure S2. Assay the enzymatic activity of AL and S in cell extracts of mixotrophic cultures of bl. tepidum with acetate supplied. The enzymatic assays of AL were performed as described earlier (1), and oxaloacetate formation via citrate lyase was coupled to NADH oxidation catalyzed by malate dehydrogenase. The activity assays were preformed in.1 N triethanolamine Hl at ph 8.. The activity of AL was determined by the decrease at A 34 (shown in the Figure below). The activity of S was evaluated by two methods: one is measured by a coupling assays with malate dehydrogenase (MDH) containing NAD + (1.5 mm), L-malate (5 mm), acetyl-oa (.1 mm) and MDH (2 U) in.1 N triethanolamine Hl at ph 8.. The activity of S was determined by the increase at A 34. No activity of S in cell cultures can be concluded because clearly no increase was observed at A 34 (see Figure below); and the other approach was to use DTNB (5,5'-dithio-bis(2-nitrobenzoic acid), i.e. Ellman s reagent) for probing the formation of oa from condensation of OAA and acetyl-oa catalyzed by S (2). No activity of S can be detected by DTNB derivation, either (data not shown) AL S 1.7 A time (s) References: 1. Kim, W., and Tabita, F. R. (6) Journal of bacteriology 188(18), Leek, B. T., Mudaliar, S. R., Henry, R., Mathieu-ostello, O., and Richardson, R. S. (1) American journal of physiology 2(2), R

7 Figure S3. The predicted labeling pattern using [1-13 ]pyruvate (A) and [2-13 ]acetate/[3-13 ] pyruvate (B) in the OTA (blue curve) vs. RTA (red curve) cycle. The predicted labeled carbon in the OTA and RTA cycle using [1-13 ]pyruvate (A) or [2-13 ]acetate/[3-13 ] pyruvate (B) is shown in blue and red, respectively. A H 2 O PO 3 = O PEP O H 3 O pyruvate O 2 O 2 O 2 H 3 OSoA H 3 O OAA O HOO O HOO O HOO O oxidative & reductive TA cycle O 2 H O HOO 2 H HOO O 2 HOO O 2 H O 2 O 2 H O 2 H HOO O α-kg O O 2 O 2 HOO OSoA HOO Glu NH 2 O 2 H B H 2 O PO 3 = O PEP O H 3 O pyruvate O 2 O 2 O 2 H 3 OSoA H 3 O OAA O HOO O HOO O HOO O oxidative & reductive TA cycle O 2 H HOO O 2 H HOO O 2 HOO O 2 H O 2 O 2 H O 2 H HOO O α-kg O O 2 O 2 HOO OSoA HOO Glu NH 2 O 2 H 7

Tricarboxylic Acid Cycle. TCA Cycle; Krebs Cycle; Citric Acid Cycle

Tricarboxylic Acid Cycle. TCA Cycle; Krebs Cycle; Citric Acid Cycle Tricarboxylic Acid ycle TA ycle; Krebs ycle; itric Acid ycle The Bridging Step: Pyruvate D hase O H 3 - - pyruvate O O - NAD + oash O 2 NADH O H 3 - - S - oa acetyl oa Pyruvate D hase omplex Multienzyme