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

Carbofuran-mineralizing strain Novosphingobium sp. KN65.2 produces the CfdJ enzyme that converts the N-methylcarbamate insecticide to carbofuran phenol. Purified CfdJ shows a remarkably low KM towards carbofuran. Together with the carbaryl hydrolase CehA of Rhizobium sp. strain AC100, CfdJ represents a new protein family with several uncharacterized bacterial members outside the proteobacteria. Although both enzymes differ by only four amino acids, CehA does not recognize carbofuran as a substrate whereas CfdJ also hydrolyzes carbaryl. None of the CfdJ amino acids that differ from CehA were shown to be silent regarding carbofuran hydrolytic activity but one particular amino acid substitution, i.e., L152 to F152, proved crucial. CfdJ is more efficient in degrading methylcarbamate pesticides with an aromatic side chain whereas CehA is more efficient in degrading the oxime carbamate nematicide oxamyl. The presence of common flanking sequences suggest that the cfdJ gene is located on a remnant of the mobile genetic element Tnceh carrying cehA. Our results suggest that these enzymes can be acquired through horizontal gene transfer and can evolve to degrade new carbamate substrates by limited amino acid substitutions. We demonstrate that a carbaryl hydrolase can gain the additional capacity to degrade carbofuran by a single nucleotide transversion.

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

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.

Niche-specification of aerobic 2,4-dichlorophenoxyacetic acid biodegradation by tfd-carrying bacteria in the rice paddy ecosystem.

This study aimed for a better understanding of the niche specification of bacteria carrying the tfd-genes for aerobic 2,4-dichlorphenoxyacetic acid (2,4-D) degradation in the rice paddy ecosystem. To achieve this, a dedicated microcosm experiment was set up to mimic the rice paddy system, with and without 2,4-D addition, allowing spatial sampling of the different rice paddy compartments and niches, i.e., the main anaerobic bulk soil and the aerobic surface water, surface soil, root surface and rhizosphere compartments. No effect of 2,4-D on the growth and morphology of the rice plant was noted. 2,4-D removal was faster in the upper soil layers compared to the deeper layers and was more rapid after the second 2,4-D addition compared to the first. Moreover, higher relative abundances of the 2,4-D catabolic gene tfdA and of the mobile genetic elements IncP-1 and IS1071 reported to carry the tfd-genes, were observed in surface water and surface soil when 2,4-D was added. tfdA was also detected in the root surface and rhizosphere compartment but without response to 2,4-D addition. While analysis of the bacterial community composition using high-throughput 16S rRNA gene amplicon sequencing did not reveal expected tfd-carrying taxa, subtle community changes linked with 2,4-D treatment and the presence of the plant were observed. These findings suggest (i) that the surface soil and surface water are the primary and most favorable compartements/niches for tfd-mediated aerobic 2,4-D biodegradation and (ii) that the community structure in the 2,4-D treated rice paddy ecosystem is determined by a niche-dependent complex interplay between the effects of the plant and of 2,4-D.

Intra- and inter-field diversity of 2,4-dichlorophenoxyacetic acid-degradative plasmids and their tfd catabolic genes in rice fields of the Mekong delta in Vietnam.

The tfd genes mediating degradation of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) differ in composition and organization in bacterial isolates from different geographical origin and are carried by different types of mobile genetic elements (MGE). It is not known whether such global diversity of 2,4-D-catabolic MGE and their tfd gene cargo is reflected in the diversity at field scale. The genomic context of the 2,4-D catabolic genes of 2,4-D-degrading isolates from two rice fields with a 2,4-D application history, located in two distant provinces of the Vietnam Mekong delta, was compared. All isolates were ß-proteobacteria, were unique for each rice field and carried the catabolic genes on MGE and especially plasmids. Most plasmids were IncP-1ß plasmids and carried tfd clusters highly similar to those of the IncP-1ß plasmid pJP4, typified by two chlorophenol catabolic gene modules (tfd-I and tfd-II). IncP-1ß plasmids from the same field showed small deletions and/or insertions in accessory metabolic genes. One plasmid belonged to an unclassified plasmid group and carries a copy of both tfdA and tfd-II identical to those in the IncP-1ß plasmids. Our results indicate intra-field evolution and inter-field exchange of 2,4-D-catabolic IncP-1ß plasmids as well as the exchange of tfd genes between different plasmids within a confined local environment.

Draft Genome Sequence of the Carbofuran-Mineralizing Novosphingobium sp. Strain KN65.2.

Complete mineralization of the N-methylcarbamate insecticide carbofuran, including mineralization of the aromatic moiety, appears to be confined to sphingomonad isolates. Here, we report the first draft genome sequence of such a sphingomonad strain, i.e., Novosphingobium sp. KN65.2, isolated from carbofuran-exposed agricultural soil in Vietnam.