Molecular and ecological characteristics of carbamate pesticide degrading bacteria

Abstract

Carbamate insecticides, such as carbofuran and fenobucarb, have been widely used to control a variety of pests throughout the world. These insecticides are seldom degraded in soils and have been shown to have toxicity to animals and microorganisms. Hence several studies have focused on the isolation and characterization of carbamate insecticide-degrading microorganisms. In spite of these studies, little information on pathways and genes involved in degradation of carbamate insecticides is available. In this study, Novosphingobium sp. 31B isolated from agricultural soil was observed to degrade fenobucarb through 2-sec-butylphenol and 3-sec-butylcatechol. This is the first report of degradative pathway of fenobucarb. In addition, Sphingobium sp. JE1 was observed to degrade carbofuran through a new pathway, via carbofuran-phenol and 5-hydroxycarbofuran. Sphingobium sp. JE1 had a carbofuran-degradative 220 kb-sized plasmid pJE1 with 229 ORFs and novel catabolic genes. In particular, the transfer-related gene order in the operon was remarkably homologous in carbofuran-degrading bacteria. To determine the interaction between expression of transfer-related genes and degradation of insecticide, quantitative real time PCR analysis was performed. One of the homologous genes, ORF189, was almost relatively four-fold expressed when carbofuran was used as the sole carbon source compared to when glucose was used as the sole carbon source. This results showed that ORF189 may be associated with carbofuran degradation. To identify the genetically-related site of carbofuran degradation in plasmid, the wild type strain JE1 was mutated using the suicide vector pSUP5011. Seven mutated strains could not degrade carbofuran

these mutants of strain JE1 were each inserted with the Tn5 sequence in plasmid. This indicates that carbofuran-degradation related genes are located in plasmid. Furthermore, changes in utilization patterns of substrate related to carbamate pesticide on seven mutants indicates that the site of Tn5 insertion is not only related the carbofuran but also other carbamate pesticide degradation. In addition, a methyl parathion-degrading bacterium 5N26T was isolated from an agricultural soil cultivated with Chinese cabbage (Brassica campestris L.). Moreover, this strain was a taxonomically novel species of the genus Roseomonas. During the incubation of strain 5N26T in mineral medium containing 50 ppm of methyl parathion as sole carbon source, methyl parathion was converted to 4-nitrophenol. Another methyl parathion-degrading bacterial strain 5G38T was isolated. The novel strain can be distinguished from the other species of the genus Pedobacter by physiological properties. The name Pedobacter namyangjuensis sp. nov. is therefore proposed for strain 5G38T as the type strain. In this study, carbamate insecticide-degradative pathways of Sphingobium sp. and Novosphingobium sp. were characterized, nucleotide sequence of carbofuran degradative plasmid pJE1 was analyzed, and carbofuran degradation-related gene of pJE1, ORF189, was identified. In addition, another pesticide methyl parathion degrading bacteria were proposed as novel species within the genera Roseomonas and Pedobacter.