Sequence Analysis of the Genes Involved in the meta-Cleavage Pathway of a PCB Degrading Thermophile
Gouri MUKERJEE-DHAR*a, Hiroki HIRABAYASHIb, Takashi HATTAb, Minoru SHIMURAa, and Kazuhide KIMBARAa
a Environmental Biotechnology Laboratory, Railway Technical Research Institute, 2-8-38 Hikaricho, Kokubunji, Tokyo 185, Japan
b Research Institute of Technology, Okayama University of Science, 401-1 Seki, Okayama, Okayama 703, Japan
Bacillus sp. strain JF8, a thermophile which besides utilizing aromatic compounds like benzoic acid, biphenyl and naphthalene for growth, can also biodegrade many chlorinated congeners present in complex PCB mixtures at 60℃. Biphenyl grown cells of strain JF8 can transform most of the dichlorobiphenyls, trichlorobiphenyls and also a few tetrachlorobiphenyls present in 20μg/ml of Kaneclor 300, a PCB mixture equivalent to Aroclor 1242.
To study the molecular structure of the genes involved in the meta-cleavage of benzoic acid/biphenyl, a gene bank of 3-5 kb HindIII fragments of strain JF8 was constructed. A clone which turned yellow on being sprayed with solutions of catechol and 2,3-dihydroxybiphenyl, contained a 4 kb HindIII insert. The upstream and downstream regions of the HindIII fragment containing the gene coding for the meta-cleavage enzyme were cloned and sequenced. Sequencing revealed 60RFs in the order ORF 1, ORF 2, ORF 3, ORF 4, ORF 5 and ORF 6.
The deduced amino acid sequence of ORF 1, which is situated 1 kb upstream of ORF 2, exhibits about 35% identity to BphA1, the large subunit of the multicomponent biphenyl dioxygenase enzyme of Rhodococcus sp. strain RHA1 and Pseudomonas sp. strain KKS 102. The deduced amino acid sequence of ORF 2, which is just upstream of ORF 3, revealed a 47% and 44% identity to BphB (2,3-dihydrodiol dehydrogenase) of Rhodococcus sp. strain RHA 1 and Pseudomonas sp. strain KKS 102, respectively. The deduced amino acid sequence of ORF 3, which codes for the meta-cleavage enzyme exhibited a 36% identity to PheB (catechol 2,3- dioxygenase) of B. stearothermophilus FDTP-3 and a 34% identity to Cdo (catechol 2,3-dioxygenase) of Rhodococcus rhodochorous CTM.
Three closely placed cistrons, ORF 4, ORF 5 and ORF 6 are located downstream of ORF 3. The deduced amino acid sequences of these ORFs exhibit about 50% identity to the polypeptides involved in the meta-cleavage pathway of the phenol degrading Pseudomonas sp. strain CF600 (Dmp E, DmpF, and DmpG) and enzymes of the lower biphenyl pathway of Pseudomonas sp. strain KKS 102 (BphE, BphG and BphF). This study reveals that the gene order and deduced amino acid sequence of meta-cleavage pathway genes in the thermophile, strain JF8, are similar to meta- cleavage pathway genes of mesophiles.
