Isolation and characterization of an N-methylcarbamate insecticide-degrading methylotrophic bacterium

Abstract

A gram-negative bacterium which hydrolyzed aryl N-methylcarbamate insecticides was isolated from an agricultural soil which quickly degraded these pesticides. This organism, designated strain ER2, grew on carbofuran as a sole source of carbon and nitrogen with a doubling time of 3 h in a mineral salts medium. The aromatic nucleus of the molecule was not metabolized, and carbofuran 7-phenol accumulated as the end product of metabolism. The insecticides carbaryl, bendiocarb, and propoxur were similarly hydrolyzed, with each yielding the corresponding phenol. Strain ER2 contained two plasmids (120 and 130 kb). A probe cloned from the pDL11 plasmid ofAchromobacter sp. strain WM111, which encodes the carbofuran hydrolase (mcd) gene (P. H. Tomasek and J. S. Karns, J. Bacteriol. 171:4038-4044, 1989), hybridized to the 120-kb plasmid. Restriction fragment profiles of pDLII and strain ER2 plasmid DNAs suggested that the 120-kb plasmid of strain ER2 is very similar to pDL11. On the basis of the results of biochemical tests, 16S rRNA sequence analysis, and membrane lipid analyses, strain ER2 was found to be a phylogeneticaily unique type II methylotroph. The constitutive carbofuran hydrolase activity in glucose-grown cells increased sevenfold when strain ER2 was grown in the presence of 100 mg of carbofuran per liter as the sole source of carbon and nitrogen or as the sole nitrogen source in the presence of glucose. Growth on carbofuran resulted in the induction of enzymes required for methylamine-dependent respiration and the serine pathway of formaldehyde assimilation. These results indicate that the carbofuran hydrolase mcd gene is conserved on a plasmid found in organisms from different geographic areas and that the specific activity of carbofuran degradation may increase in response to carbofuran treatment.

Identification and characterization of trimethylamine N‐oxide (TMAO) demethylase and TMAO permease in Methylocella silvestris BL2

“…Several processes contribute to biological MA production in the terrestrial environment, including degradation of herbicides and pesticides (Topp et al, 1993;Kamanavalli and Ninnekar, 2000;Bhadbhade et al, 2002), protein putrefaction (Kamiya and Ose, 1984), anaerobic microbial respiration (Barrett and Kwan, 1985) as well as degradation of quaternary amines (King, 1988). Quaternary amines such as choline and carnitine are significant components of eukaryotic cells and are released to the environment because of normal cell turnover and programmed cell death.…”

Genetic diversity of carbofuran-degrading soil bacteria

“…Homology research and sequence comparisons were achieved on the Infobiogen server (Villejuif, France) with Fasta version 3.3t04 [21] and Align version 2.0u [22] programs. Reference strains of carbofuran-degrading bacteria, Sphingomonas CF06 [23], WM111 [5] and ER2 [7] were included in the phylogenetic tree as well as some reference methylotrophic strains [24].…”

“…Isolates from French soils are designated by a capital letter followed by a subscripted number: C and D refer, respectively, to the ¢rst series of isolates [8] and the new series isolated in this study, the subscripted number corresponds to the French department or district where the soil was collected from. WM111 and ER2 are reference strains [5,7]. c Isolates in bold are representative for each 16S ribotype whose 16S rRNA gene was partially sequenced.…”

“…Carbofuran-degrading micro-organisms generally utilize methylamine for growth and thus belong to the heterogeneous group of bacteria known as facultative methylotrophs. Several reports have stressed the high degree of diversity among these organisms which can belong to the K-, L-and Q-subdivisions of the Proteobacteria [7,24]. Fig.…”

Isolation and characterization of 23 carbofuran-degrading bacteria from soils from distant geographical areas

“…They then suggested that a comparative analysis of conserved gene sequences (16S rRNA) would have been the method of choice to characterize the bacterial isolates with high certainty. This has been done by Topp et al (1993) but for only one isolate. The objectives of this study were to isolate micro-organisms with the ability to rapidly degrade carbofuran from soils from UK and Greece, and to characterize them using molecular methods.…”