Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in gram-negative bacteria.
A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase.
Tomasek
P H
PH
Pesticide Degradation Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705.
Karns
J S
JS
D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001673:Biodegradation, Environmental; D015153:Blotting, Western; D002235:Carbofuran / Q000378:metabolism*; D003001:Cloning, Molecular; D003360:Cosmids; D004274:DNA, Recombinant / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism; D007306:Insecticides / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D015183:Restriction Mapping
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D007306:Insecticides; D006867:Hydrolases; D002235:Carbofuran