GENES FOR 2,4,5-TRICHLOROPHENOXYACETIC ACID METABOLISM IN BURKHOLDERIA-CEPACIA AC1100 - CHARACTERIZATION OF THE TFTC AND TFTD GENES AND LOCATIONS OF THE TFT OPERONS ON MULTIPLE REPLICONS

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4 ,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon an d energy. The enzyme which converts the first intermediate in the path way, 2,4,5-trichlosophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftD genes (48). A degenerate printer was designed from the N terminu s of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes hom a genomic library of AC1100. The derived amin o acid sequences of tftC and tftD show significant homology to the two -component monooxygenases HadA of Burkholderia pickettii, HpaBC of Esc herichia coli, and HpaAH of Klebsiella pneumonia. Expression of the tf tC and tftD genes appeared to be induced when they were grown in the p resence of 2,4,5-T, as shown by RNA slot blot and primer extension ana lyses. Three sets of cloned tft genes were used as probes to explore t he genomic organization of the pathway. Pulsed-field gel electrophores is analyses of whole chromosomes of B. cepacia AC1100 demonstrated tha t the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, a nd 0.15 Mbp, designated I to V, respectively. The tft genes are locate d on the smaller replicons: the tftAB cluster is on replicon IV, tftEF GH is on replicon III, and copies of the tjtC and the tftCD operons ar e found on both replicons III and IV. When cells were grown in the abs ence of 2,4,5-T, the genes were Lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In o ne mutant, the tftA and tftB genes translocated from one replicon to a nother, with the concomitant loss of tftEFGH and one copy of tftCD.