MOLECULAR CHARACTERIZATION OF THE CAI OPERON NECESSARY FOR CARNITINE METABOLISM IN ESCHERICHIA-COLI

The sequence encompassing the cai genes of Escherichia coli, which enc ode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five addit ional open reading frames were identified. They belong to the caiTABCD E operon, which was shown to be located at the first minute on the chr omosome and transcribed during anaerobic growth in the presence of car nitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a function al H-NS protein, in relation to the consensus sequences detected in th e promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicte d molecular masses of 56 613 Da (CaiT), 42 564 Da (CaiA), 59 311 Da (C aiC), 32 329 Da (CaiD) and 21 930 Da (CaiE). Amino acid sequence simil arity or enzymatic analysis supported the function assigned to each pr otein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/car nitine CoA ligase. CaiD bears strong homology with enoyl hydratases/is omerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the bas al level of carnitine dehydratase activity. It is inferred that CaiE i s an enzyme involved in the synthesis or the activation of the still u nknown cofactor required for carnitine dehydratase and carnitine racem ase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between A grobacterium and Rhizobium.