Cloning, physical mapping and expression of chromosomal genes specifying degradation of the herbicide 2,4,5-T by Pseudomonas cepacia AC1100.
A genomic library of total DNA of Pseudomonas cepacia AC1100 was constructed on a broad-host-range cosmid vector pCP13 in Escherichia coli AC80. A 25-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. This 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. Soc. Environ. Toxicol. Chem. USA 8 (1987) 127]. A simple 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. Subcloning 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. Both fragments are identified to be chromosomal in origin. Hybridization 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. The cloned fragments did not show homology to plasmid DNAs carrying degradative genes for toluene, naphthalene and 3-chlorobenzoate.
Sangodkar
U M
UM
Department of Microbiology and Immunology, University of Illinois, College of Medicine, Chicago 60612.
Chapman
P J
PJ
Chakrabarty
A M
AM
D015085:2,4,5-Trichlorophenoxyacetic Acid / Q000493:pharmacokinetics*; D015139:Blotting, Southern; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003470:Culture Media; D011088:DNA Ligases / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008658:Inactivation, Metabolic; D009154:Mutation; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D003470:Culture Media; D015085:2,4,5-Trichlorophenoxyacetic Acid; D011088:DNA Ligases