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Appl Environ Microbiol, June 1998, p. 2086-2093, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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
Anette
Hübner,1
Clyde E.
Danganan,1
Luying
Xun,2
A. M.
Chakrabarty,1 and
William
Hendrickson1,*
Department of Microbiology and Immunology,
College of Medicine, University of Illinois at Chicago, Chicago,
Illinois 60612,1 and
Department of
Microbiology, Washington State University at Tri-Cities, Richland,
Washington 993522
Received 22 December 1997/Accepted 26 March 1998
Burkholderia cepacia AC1100 uses the chlorinated
aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole
source of carbon and energy. The enzyme which converts the first
intermediate in the pathway, 2,4,5-trichlorophenol, 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 primer was designed from the N
terminus of the 58-kDa polypeptide and used to isolate a clone
containing the tftC and tftD genes from a
genomic library of AC1100. The derived amino acid sequences of
tftC and tftD show significant homology to the
two-component monooxygenases HadA of Burkholderia
pickettii, HpaBC of Escherichia coli, and HpaAH of
Klebsiella pneumonia. Expression of the tftC
and tftD genes appeared to be induced when they were grown
in the presence of 2,4,5-T, as shown by RNA slot blot and primer
extension analyses. Three sets of cloned tft genes were
used as probes to explore the genomic organization of the pathway.
Pulsed-field gel electrophoresis analyses of whole chromosomes of
B. cepacia AC1100 demonstrated that the genome is
comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp,
designated I to V, respectively. The tft genes are located
on the smaller replicons: the tftAB cluster is on replicon
IV, tftEFGH is on replicon III, and copies of the
tftC and the tftCD operons are found on both
replicons III and IV. When cells were grown in the absence 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 one mutant, the
tftA and tftB genes translocated from one
replicon to another, with the concomitant loss of tftEFGH
and one copy of tftCD.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, College of Medicine, University of
Illinois at Chicago, 835 S. Wolcott, Chicago, IL 60612. Phone: (312)
996-5600. Fax: (312) 996-6415. E-mail: whend{at}uic.edu.
Appl Environ Microbiol, June 1998, p. 2086-2093, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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