Research Product

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an eleven step pathway through p-cumate (p-isoproylbenzoate) to isobutyrate, pyruvate, and acetyl-coenzyme A. The cym operon, encoding the conversion of p-cymene to p -cumate, is located just upstream of the cmt operon which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P.putida F1; the sequences of an 11,792 base pair DNA segment carrying the cym operon and a 2,679 base pair DNA segment connecting the cmt and tod operons are described here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p -cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehyldrogenase; cymD encodes a putative outer membrane protein that is related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; cymE encodes an acetyl-coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. Studies of recombinant bacteria carrying either the operator-promoter region of the cym or the cmt operon upstream of genes encoding assay-able enzymes, in the presence or absence of cymR, demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Upstream of cymR and between the cmt and tod operons are short (less than 350 base pairs) DNA segments having a high degree of sequence homology to each other; these may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P.putida F1.