Acetogenic bacteria fix CO or CO2 by a pathway of autotrophic growth called the acetyl-CoA (or Wood) pathway. Key enzymes in the pathway are a methyltransferase, a corrinoid/Fe-S protein, a disulfide reductase, and a carbon monoxide dehydrogenase. This manuscript describes the isolation of the genes that code for the methyltransferase, the two subunits of the corrinoid/Fe-S protein, and the two subunits of carbon monoxide dehydrogenase. These five genes were found to be clustered within an 10-kilobase segment on the Clostridium thermoaceticum genome. The proteins were expressed at up to 5-10% of Escherichia coli cell protein, and isopropyl .8-D-thiogalactopyranoside had no effect on the levels of expression, implying that the C. thermoaceticum inserts contained transcriptional and translational signals that were recognized by E. coli. The methyltransferase is expressed in E. coli in a fully active dimeric form with a specific activity and heat stability similar to the enzyme expressed in C. thermoaceticum. However, both the corrinoid/Fe-S protein and carbon dioxide dehydrogenase, although expressed in high amounts and with identical subunit molecular weights in E. coli, are inactive and less heat stable than are the native enzymes from C. thermoaceticum. sulfides, and 1-3 Zn atoms per dimer (7). The roles of CODH in the pathway are to bind CO (9, 10), a methyl group (11), and CoA (12, 13) and to catalyze the actual synthesis of acetyl-CoA (12).In this manuscript, we report the cloning of the genes for CODH, MeTr, and C/Fe-SP and the expression of the proteins at high levels in Escherichia coli in the absence of any inducer. We have established that these genes are clustered within a 10-kilobase (kb) DNA segment in the C. thermoaceticum genome and that the CODH genes are directly upstream of the 55-kDa subunit of the C/Fe-SP. We suggest that these clustered genes contain promoter-like sequences and translational signals that are recognized by E. coli. MeTr is expressed in E. coli as a heat-stable dimer and is fully active. Both the C/Fe-SP and CODH, although expressed in high amounts by E. coli, are inactive and are much less heat stable than are the active enzymes from C. thermoaceticum.A preliminary report describing the cloning of the C/Fe-SP and MeTr-encoding genes has been published (14). MATERIALS AND METHODSBacterial Strains, Plasmids, and Growth Conditions. C. thermoaceticum, DSM 521, was cultured in 20-liter carboys at 55°C under CO2 as described by Ljungdahl and Andreesen (15). E. coli K-12 strain JM109 (F' traD36 proAB laclqZAM-15/supE44 thi) was cultured on SOB medium (16). Colonies transformed with pUC9 (17) were grown at pH 7.5 at 37°C on LB-ampicillin medium (16) containing 0.8% tryptone, 0.5% yeast extract, 0.5% sodium chloride, and ampicillin at 0.1 mg/ml. 5-Bromo-4-chloro-3-indolyl (3-D-galactopyranoside and isopropyl B-D-thiogalactopyranoside (IPTG) (both from Boehringer Mannheim) were added to the LB-ampicillin medium to final concentrations of 0.032% and 1.6 mM, respectively, for colo...