METHOD FOR ISOLATION OF AUXOTROPHS IN THE METHANOGENIC ARCHAEBACTERIA - ROLE OF THE ACETYL-COA PATHWAY OF AUTOTROPHIC CO2 FIXATION IN METHANOCOCCUS-MARIPALUDIS

A procedure was developed for the enrichment of auxotrophs in the antibiotic-insensitive archaebacterium Methanococcus. After mutagenesis with ethyl methane-sulfonate, growing cells were selectively killed upon exposure to the base analogs 6-azauracil and 8-azahypoxanthine for 48 hr. Using this method, eight independent acetate auxotrophs of Methanococcus maripaludis were isolated. Six of the auxotrophs had an absolute growth requirement for acetate and contained 1-16% of the wild-type levels of CO dehydrogenase. Three of these six also contained 14-29% of the wild-type levels of pyruvate oxidoreductase and 12-30% of the wild-type levels of pyruvate synthase. Two spontaneous revertants of these latter auxotrophs regained the ability to grow normally in the absence of acetate and wild-type levels of CO dehydrogenase, acetyl-CoA synthase, pyruvate oxidoreductase, and pyruvate synthase. Likewise, a spontaneous revertant of an auxotroph with reduced levels of CO dehydrogenase and wild-type levels of pyruvate oxidoreductase regained the ability to grow normally in the absence of acetate and wild-type levels of CO dehydrogenase and acetyl-CoA synthase. Two additional auxotrophs grew poorly in the absence of acetate but contained wild-type levels of CO dehydrogenase and pyruvate oxidoreductase. These results provide direct genetic evidence for the Ljungdahl-Wood pathway [Ljungdahl, L. G. (1986) Annu. Rev. Microbiol. 40, 415-450; Wood, H. G., Ragsdale, S. W. & Pezacka, E. (1986) Trends Biochem. Sci. 11, 14-18] of autotrophic acetyl-CoA biosynthesis in the methanogenic archaebacteria. Moreover, it suggests that the acetyl-CoA and pyruvate synthases may share a common protein or coenzyme component, be linked genetically, or be regulated by a common system.