CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION OF THE ESCHERICHIA-COLI GENE ENCODING CARNITINE DEHYDRATASE

Carnitine dehydratase from Escherichia coli O44 K74 is an inducible en zyme detectable in cells grown anaerobically in the presence of L-(-)- carnitine or crotonobetaine. The purified enzyme catalyzes the dehydra tion of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, en coding carnitine dehydratase, was isolated by oligonucleotide screenin g from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M( r) of 45,074. The identity of the gene product was first assessed by i ts comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzy matic activity. Moreover, the N-terminal amino acid sequence of the pu rified protein was found to be identical to that predicted from the ge ne sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be t he gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.