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Journal of Bacteriology, February 2000, p. 937-943, Vol. 182, No. 4
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Set of Genes Encoding a Second Toluene Efflux
System in Pseudomonas putida DOT-T1E Is Linked to the
tod Genes for Toluene Metabolism
Gilberto
Mosqueda and
Juan-Luis
Ramos*
Department of Biochemistry and Molecular
Biology of Plants, Estación Experimental del Zaidín,
Consejo Superior de Investigaciones Científicas, E-18008
Granada, Spain
Received 30 July 1999/Accepted 11 November 1999
Sequence analysis in Pseudomonas putida DOT-T1E
revealed a second toluene efflux system for toluene metabolism encoded
by the ttgDEF genes, which are adjacent to the
tod genes. The ttgDEF genes were expressed in
response to the presence of aromatic hydrocarbons such as toluene and
styrene in the culture medium. To characterize the contribution of the
TtgDEF system to toluene tolerance in P. putida,
site-directed mutagenesis was used to knock out the gene in the
wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This
mutant carried a Tn5 insertion in the ttgABC gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out
the ttgD gene and encoding resistance to tellurite was
constructed in vitro and transferred to the corresponding host
chromosome via the suicide plasmid pKNG101. Successful replacement of
the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single ttgD mutant, DOT-T1E-1,
and a double mutant with knock outs in the ttgD and
ttgA genes, DOT-T1E-82, were obtained and characterized for
toluene tolerance. This was assayed by the sudden addition of toluene
(0.3% [vol/vol]) to the liquid culture medium of cells growing on
Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene
supplied via the gas phase (induced). Induced cells of the single
ttgD mutant were more sensitive to sudden toluene shock
than were the wild-type cells; however, noninduced wild-type and
ttgD mutant cells were equally tolerant to toluene shock.
Noninduced cells of the double DOT-T1E-82 mutant did not survive upon
sudden toluene shock; however, they still remained viable upon sudden
toluene shock if they had been previously induced. These results are
discussed in the context of the use of multiple efflux pumps involved
in solvent tolerance in P. putida DOT-T1E.
*
Corresponding author. Mailing address:
CSIC-Estación Experimental del Zaidín, Prof. Albareda 1, E-18008 Granada, Spain. Phone: 34-958-121011. Fax: 34-958-129600. E-mail: jlramos{at}eez.csic.es.
Journal of Bacteriology, February 2000, p. 937-943, Vol. 182, No. 4
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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