Pseudomonas putida CE2010 can degrade biphenyl by a mosaic pathway encoded by the tod operon and cmtE, which are identical to those of P. putida F1 except for a single base difference in the operator-promoter region of the cmt operon

Laboratory of Microbiology, RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan 1 Author for correspondence: Toshiaki Kudo. Tel: +81 48 467 9544. Fax: +81 48 462 4672. e-mail: tkudo{at}postman.riken.go.jp Psudomonas putida CE2010 can assimilate biphenyl despite its high similarity to P. putida F1. Biphenyl degradation in strain CE2010 was achieved using a mosaic of pathways consisting of the cmt and tod operons. CmtE hydrolysed 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta -cleavage product of 2,3-dihydroxybiphenyl. This enzyme was expressed differently in strains CE2010 and F1. A cmtE disruption mutant, a tod operon disruption mutant and a cmt operon disruption mutant were unable to utilize biphenyl. The introduction of the cmtE gene enabled the cmt operon disruption mutant to grow on biphenyl. A single base difference was found in the cmt promoter–operator region in strain CE2010, compared with that of strain F1. CymR protein was purified from Escherichia coli and binding assays were performed, the results of which suggested that the protein bound less strongly to the CE2010 operator sequence than to the F1 operator sequence. Exchanging the F1 promoter–operator fragment into strain CE2010 resulted in a loss of biphenyl degradation capacity. These results indicate that cmtE is not effectively repressed by CymR in strain CE2010, leading to low constitutive expression and, therefore, low growth on biphenyl. Keywords: biphenyl, mosaic pathway, biodegradation, Pseudomonas putida Abbreviations: 2,3-DHBP, 2,3-dihydroxybiphenyl; 2,3-DHBD, 2,3-dihydroxybiphenyl dioxygenase; HPDA, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid; HOHD, 2-hydroxy-6-oxo-hepta-2,4-dienoic acid; Ap, ampicillin; Cb, carbenicillin; Km, kanamycin; Tc, tetracycline The DDBJ accession numbers for the sequences reported in this paper are AB042508 and AB042509.