NUCLEOTIDE-SEQUENCE AND FUNCTIONAL-ANALYSIS OF THE GENES ENCODING 2,4,5-TRICHLOROPHENOXYACETIC ACID OXYGENASE IN PSEUDOMONAS-CEPACIA AC1100

Pseudomonas cepacia AC1100 is able to use the chlorinated aromatic com pound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as the sole source o f carbon and energy. One of the early steps in this pathway is the con version of 2,4,5-T to 2,4,5-trichlorophenol (2,4,5-TCP). 2,4,5-TCP acc umulates in the culture medium when AC1100 is grown in the presence of 2,4,5-T. A DNA region from the AC1100 genome has been subcloned as a 2.7-kb SstI-XbaI DNA fragment, which on transfer to Pseudomonas aerogi nosa PAO1 allows the conversion of 2,4,5-T to 2,4,5-TCP. We have deter mined the directions of transcription of these genes as well as the co mplete nucleotide sequences of the genes and the number and sizes of t he polypeptides synthesized by pulse labeling experiments. This 2.7-kb DNA fragment encodes two polypeptides with calculated molecular masse s of 51 and 18 kDa. Proteins of similar sizes were seen in the T7 puls e-labeling experiment in Escherichia coli. We have designated the gene s for these proteins tftA1 (which encodes the 51-kDa protein) and tftA 2 (which encodes the 18-kDa protein). TftA1 and TftA2 have strong amin o acid sequence homology to BenA and BenB from the benzoate 1,2-dioxyg enase system of Acinetobacter calcoaceticus, as well as to XylX and Xy lY from the toluate 1,2-dioxygenase system of Pseudomonas putida. The Pseudomonas aeruginosa PAO1 strain containing the 2.7-kb SstI-XbaI fra gment was able to convert not only 2,4,5-T to 2,4,5-TCP but also 2,4-d ichlorophenoxyacetic acid to 2,4-dichlorophenol and phenoxyacetate to phenol.