Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13.
DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid. Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp. B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate .
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D006846:Hydrocarbons, Halogenated / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics*; D010105:Oxygenases / Q000235:genetics*; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity
D006846:Hydrocarbons, Halogenated; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D010105:Oxygenases; C023172:toluene dioxygenase; C022619:salicylate 1-monooxygenase; C025312:3,5-cyclohexadiene-1,2-diol-1-carboxylate dehydrogenase; D004262:DNA Restriction Enzymes