Genetic and metabolic analysis of the carbofuran catabolic pathway in Novosphingobium sp. KN65.2

Abstract

The widespread agricultural application of carbofuran and concomitant contamination of surface and ground waters has raised health concerns due to the reported toxic effects of this insecticide and its degradation products. Most bacteria that degrade carbofuran only perform partial degradation involving carbamate hydrolysis without breakdown of the resulting phenolic metabolite. The capacity to mineralize carbofuran beyond the benzofuran ring has been reported for some bacterial strains, especially sphingomonads, and some common metabolites, including carbofuran phenol, were identified. In the current study, the catabolism of carbofuran by Novosphingobium sp. KN65.2 (LMG 28221), a strain isolated from a carbofuran-exposed Vietnamese soil and utilizing the compound as a sole carbon and nitrogen source, was studied. Several KN65.2 plasposon mutants with diminished or abolished capacity to degrade and mineralize carbofuran were generated and characterized. Metabolic profiling of representative mutants revealed new metabolic intermediates, in addition to the initial hydrolysis product carbofuran phenol. The promiscuous carbofuran-hydrolyzing enzyme Mcd, which is present in several bacteria lacking carbofuran ring mineralization capacity, is not encoded by the Novosphingobium sp. KN65.2 genome. An alternative hydrolase gene required for this step was not identified, but the constitutively expressed genes of the unique cfd operon, including the oxygenase genes cfdC and cfdE, could be linked to further degradation of the phenolic metabolite. A third involved oxygenase gene, cfdI, and the transporter gene cftA, encoding a TonB-dependent outer membrane receptor with potential regulatory function, are located outside the cfd cluster. This study has revealed the first dedicated carbofuran catabolic genes and provides insight in the early steps of benzofuran ring degradation.

Expanded insecticide catabolic activity gained by a single nucleotide substitution in a bacterial carbamate hydrolase gene

“…Degradation by micro-organisms is an important route for carbofuran dissipation in treated soils. Carbofuran-degrading bacteria have been isolated from various geographically separated areas of the world from soils that were regularly treated with the pesticide (Chaudhry and Ali, 1988;Desaint et al, 2000;Yan et al, 2007;Nguyen et al, 2014;Shin et al, 2012Shin et al, , 2014. The Mcd enzyme catalyses the hydrolysis of carbofuran in Achromobacter sp.…”

“…Mineralization experiments with 14 C-aromatic-ring-labeled carbofuran demonstrated the ability of Novosphingobium sp. KN65.2 to degrade the aromatic ring of carbofuran and use it a carbon source (Nguyen et al, 2014). Strain KN65.2 metabolizes carbofuran via a unique operon consisting of the cfd genes.…”

“…Novosphingobium sp. strain KN65.2 was isolated from carbofuran‐exposed agricultural soil in Mekong Delta, Vietnam (Nguyen et al ., ). This strain, like other reported carbofuran‐degrading sphingomonads (Kim et al ., ; Yan et al ., ), utilizes carbofuran as sole carbon source with carbofuran phenol as a major intermediate.…”

Engineering Pseudomonas putida KT2440 for simultaneous degradation of carbofuran and chlorpyrifos

“…Both carbofuran and CP were degraded quickly at the first 12 h, which accounted for 60% and 75% of the amount of the initially added pesticides respectively. The reduction in the degradation rate after 12 h may be due to the accumulation of carbonfuran phenol and TCP, which have antimicrobial activity and are toxic to the bacterial growth and metabolism (Singh et al ., ; Nguyen et al ., ). In contrast, no decrease in the concentration of carbofuran and CP was detected in M9 minimal medium inoculated with P. putida KTU (data not shown).…”

“…Carbofuran is a broad‐spectrum carbamate insecticide that is used widely for disease control in vegetable, fruit and crops. However, the potential health risks to humans posed by excessive use of carbofuran are not ignored because carbofuran is a potent AChE inhibitor (Nguyen et al ., ). Carbofuran and CP are often applied to agricultural soil together, thus, combined pollution caused by them needs to be resolved by developing new techniques (Rama Krishna and Philip, ).…”