A NOVEL ALTERNATE ANAPLEROTIC PATHWAY TO THE GLYOXYLATE CYCLE IN STREPTOMYCETES

ccr encoding crotonyl coenzyme A (CoA) reductase (CCR), which catalyze s the conversion of crotonyl-CoA to butyryl-CoA in the presence of NAD PH, was previously cloned from Streptomyces collinus. We now report th at a complete open reading frame, designated meaA, is located downstre am from ccr. The predicted gene product showed 35% identity with methy lmalonyl-CoA mutases from various sources. In addition, the predicted amino acid sequences of S. collinus ccr and meaA exhibit strong simila rity to that of adhA (43% identify), a putative alcohol dehydrogenase gene, and meaA (62% identity) of Methylobacterium extorquens, respecti vely. Both adhA and meaA are involved in the assimilation of C-1 and C -2 compounds in an unknown pathway in the isocitrate lyase (ICL)-negat ive Methylobacterium. We have demonstrated that S. collinus can grow w ith acetate as its sole carbon source even though there is no detectab le ICL, suggesting that in this organism ccr and meaA may also be invo lved in a pathway for the assimilation of C, compounds, Previous studi es with streptomycetes provided a precedent for a pathway that initiat es with the condensation of two acetyl-CoA molecules to form butyryl-C oA, which is then transformed to succinyl-CoA with two separate CoB12- mediated rearrangements and a series of oxidations. The biological fun ctions of ccr and meaA in this process were investigated by gene disru ption, A ccr-blocked mutant showed no detectable crotonyl-Coil reducta se activity and, compared to the wild-type strain, exhibited dramatica lly reduced growth when acetate was the sole carbon source, An meaA-bl ocked mutant also exhibited reduced growth on acetate, However, both m ethylmalonyl-CoA mutase and isobutyryl-CoA mutase, which catalyze the two CoB12-dependent rearrangements in this proposed pathway, were show n to be present in the meaA-blocked mutant. These results suggested th at both ccr and meaA are involved in a novel pathway for the growth of S. collinus when acetate is its sole carbon source.