Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.Carbamate insecticides such as carbaryl (1-naphthyl N-methylcarbamate) are broad-spectrum insecticides that comprise the major proportion of agricultural pesticides used in today's agricultural industry. These compounds are considered hazardous because they potently inhibit acetylcholine esterase (9) and the N-nitrosocarbamates formed are potent mutagens (8). On the other hand, these pesticides generally do not persist in soil for a long time, and the persistence of these compounds in agricultural soil is due to repeated applications (10, 38). From these perspectives, an understanding of the degradation mechanism is needed to control the persistence of these pesticides in soil.Soil microorganisms are thought to play a significant role in the reduction of pesticides in soil, and many soil bacteria capable of degrading carbamate pesticides have been isolated and characterized (4,6,16,17,21,28). The biochemical characteristics of carbamate pesticide hydrolases have also been reported (5,17,18,20,25). However, little is known about the genes for these enzymes. The mcd gene, which encodes a carbofuran hydrolase, is located on a 100-kb plasmid called pPDL11, and it has been cloned from Achromobacter sp. strain WM111 (41). This gene was shown to be present in many bacteria and to be encoded on a 100-, 105-, 115-, or 124-kb plasmid found in diverse bacteria isolated from geographically distant areas (6, 29). However, the structure of the carbamate insecticide degradative gene has not as yet been reported, although the nucleotide sequence of mcd gene is available (accession no. AF160188).The present study characterizes a carbaryl hydrolase purified from Rhizobium sp. strain AC100. From the result of a plasmid-curing experiment, it was suggested that the carbamate insecticide degradative gene is encoded on the plasmid. We then cloned the degradative gene from the plasmid DNA and determined the nucleotide sequence of the 10-kb region containing the degradative gene. The sequence analysis suggested that the g...
