Nucleotide Sequence and Genetic Structure of a Novel Carbaryl Hydrolase Gene ( cehA ) from Rhizobium sp. Strain AC100

Abstract

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...

SulE, a Sulfonylurea Herbicide De-Esterification Esterase from Hansschlegelia zhihuaiae S113

“…Esterases have played important roles in the hydrolysis of a wide range of ester-containing xenobiotics. The genes for esterases, involved in the hydrolysis of many pesticides and herbicides, such as carbaryl, carbofuran, carbendazim, phenmedipham, parathion, phenylurea, and pyrethroids, have been cloned and characterized (11,18,35,36,39,40,41). To the best of our knowledge, SulE is the first reported esterase that can hydrolyze sulfonylurea herbicides.…”

Novel Three-Component Rieske Non-Heme Iron Oxygenase System Catalyzing the N -Dealkylation of Chloroacetanilide Herbicides in Sphingomonads DC-6 and DC-2

“…IstA1 and IstB1 share 100% and 99% amino acid sequence identities to the sequences of IstA and IstB from Rhizobium sp. strain AC100, respectively (32). Downstream of cndA, there are also two genes, tnpA1 and tnpA2.…”

Cloning of a Novel Arylamidase Gene from Paracoccus sp. Strain FLN-7 That Hydrolyzes Amide Pesticides

“…have been isolated, and their microbial metabolic pathways have also been elucidated (12,22,25,32,43). To date, the sequence information for the following amide pesticide-hydrolyzing enzymes is available: carbaryl hydrolases CahA and CehA (11,12), phenmedipham hydrolase PCD (26), carbofuran hydrolase Mcd (32), carbendazimhydrolyzing esterase MheI (25), propanil-hydrolyzing amidases Amq, PamH, and PrpH (19,28,42), and phenylurea hydrolases PuhA and LibA (2,34). However, the sequence information and biochemical characterization of amidases that hydrolyze other amide pesticides, such as benzoylurea insecticides, organophosphate insecticides (dimethoate and omethoate), and sulfonylurea herbicides, have not been described.…”