FEMS Microbiology Letters70 (1990)249-254 249 Published by E|sevier FEMSLE04078 Cloning and expression of the carbon mono,,dde dehydrogenase genes from Pseudomonas thermocarboxydovorans strain C2 Gary W. Black ~, Catherine M. Lyons 2, Edwin Williams 2, John Colby ~, Michael Kehoe 2 and Catherine O'Reilly School of Biology, Sunderland Polytechnic, Sunderland and 2 Microbiology Department, The Medical School, The Unwersity, Newcastle-upon-Tyne, U.K. R~ccived2 April 1990 Ac, ccpted 5 April 1990 Key words: Carbon monoxide; Carbon monoxide dehydrogenase; Carboxydotrophic bacterium; Pseudomonas thermocarboxydovorans C2; Immunological screening; Cloning; Restriction analysis; Western blot analysis; Expression 1. SUMMARY 2. INTRODUCTION Carbon monoxide dehydrogenase (CODH) from Pseudomonas thermocarboxydovorans strain C2 is composed of three non-identical subunits. A gene library of C2 DNA in lambda vector L,,?.. was generated and screened using anti-CODH serum. Western blotting experiments revealed a protein which co-migrated with and had the same immunological reaction as the large subunit of CODH in some of the clones isolated from the library. The coding region was pinpointed to a 4 kb fragment which was subcloned into plasmid, Western blotting experiments showed that all three subunits of CODH were coded for by the sub- clone. However, no CODH activity was detected. Correspondence to: C. O'Reilly, Schoolof Biology,Sunderland Polytechnic, SunderlandSR1 35D, U.K. Bacteria capable of using CO aerobically as sole carbon and energy source are known as carboxydotrophic bacteria or simply as carboxy- dobacteria [1-4]. Carboxydobacteria are a diverse group of microorganisms and all members are able to: catalyse the oxidation of CO to CO2; transfer the electrons derived from this reaction to the respiratory electron transport chain for the generation of ATP by oxidative phosphorylation; assimilate some of the CO2 formed via the Calvin-Benson cycle; produce NAD(P)H, via re- versed electron flow, for CO~ assimilation; and withstand CO inhibition. The key enzyme in the energy metabolism of these bacteria is the molybdenum, FAD and iron-sulfur containing en- zyme, CODH (CO oxidoreduetase; CO oxidase). The carboxydobacterium Pseudomonas thermo- carboxydovorans C2, isolated from sewage [5], is capable of autotrophic growth at 45-65 ° C on CO or on a mixture of CO and hydrogen, although in 0378.1097/90/$03.50 © 1990 Federationof EuropeanMicrobiologicalSocieties