You are here:
CLONING, PHYSICAL MAPPING AND EXPRESSION OF CHROMOSOMAL GENES SPECIFYING DEGRADATION OF THE HERBICIDE 2,4,5-T BY PSEUDOMONAS CEPACIA AC100
Citation:
Sangodkar, U., A. Chakrabarty, AND P. Chapman. CLONING, PHYSICAL MAPPING AND EXPRESSION OF CHROMOSOMAL GENES SPECIFYING DEGRADATION OF THE HERBICIDE 2,4,5-T BY PSEUDOMONAS CEPACIA AC100. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-93/232.
Description:
A genomic library of total DNA of Pseudomonas Cepacia AC1100 wasconstructed on a broad-host-range cosmid vector pCP13 in Escherichia coli AC80. 5-kb segment was isolated from the library which complemented a Tn5-generated, 2,4,5-trichlorophenoxyacetic acid-negative (2,4,5-T-) mutant, P. Cepacia PT88. his mutation was partially characterized and appeared to be lacking functional enzyme required for metabolism of an intermediate of the 2,4,5-T pathway, recently identified as 5-chloro-1,2,4-trihydroxybenzene [Chapman et al., Abstr. oc. nviron. oxicol. hem. SA 8 (1987) 127]. imple colorimetric assay was developed to detect the presence of this active enzyme in intact cells and was used to determine the expression of complementing genes. ubcloning experiments showed that a 4-kb BamHI-PstI fragment and a 290-bp PstI-EcoRI fragment, separated by 1.3-kb, were required for complementation. oth fragments are identified to be chromosomal in origin. ybridization studies using the subcloned fragments revealed that in addition to a Tn5 insertion, mutant PT88 contained an extensive chromosomal deletion accounting for its 2,4,5-T phenotype. he cloned fragments did not show homology to plasmid DNAs carrying degradative genes for toluene, naphthalene and 3-chlorobenzoate.