Genetische karakterisatie van pesticide-degraderende bacteriën in de Mekong delta van Vietnam

The Mekong delta is the largest agricultural region of Vietnam. To meet the food demand for the local inhabitants and export, intensive crop cultivation results in a steady increase in application of chemical fertilizers and pesticides. Diverse chemical classes of herbicides, fungicides and insecticides have been used in the delta resulting in contamination of surface water and sediments. However, the fate of residual pesticides and capacity for microbial degradation in soils of the Mekong delta are largely unknown. It was hypothesized that the continuous selection pressure of pesticides in agricultural fields in the Mekong delta has resulted into an adapted community that is able to degrade and/or mineralize intensively used pesticides. Therefore, the overall objective of the study was to gain knowledge on the biodegradation of some currently used pesticides in the delta and the genetics involved in the catabolism of these compounds.Biodegradation and mineralization of four currently used pesticides were examined in a pristine soil and in agricultural soil samples that had a history of pesticide treatment and that were collected from two distant provinces in the Mekong delta. The examined pesticides included one herbicide, i.e., the chlorinated phenoxyacetic acid 2,4-D, and three insecticides, i.e., the chlorinated cyclodiene endosulfan, the synthetic pyrethroid cypermethrin and the methylcarbamate carbofuran. 2,4-D and cypermethrin were degraded/mineralized in all soil samples including the control soil while endosulfan was not mineralized in any of the soil samples. Carbofuran was only mineralized in soils that had been exposed to the compound. From soils showing 2,4-D degradation or carbofuran mineralization activity, ten distinct strains that use 2,4-D as sole source of carbon and energy and one strain that uses carbofuran as sole source of nitrogen, carbon and energy were isolated. The 2,4-D-degrading isolates were identified as members of the ß-proteobacterial genera Burkholderia, Cupriavidus and Ralstonia while the carbofuran-degrading strain belongs to the alfa-proteobacterial genus Novosphingobium. 2,4-D degradation in the 2,4-D-degrading isolates was plasmid encoded and the phenotype could be transferred by conjugation to a plasmid-free derivative of Cupriavidus metallidurans CH34. The plasmids were sequenced and the sequences analyzed. All plasmids contained homologues of the previously described tfd genes that code for the biodegradation of 2,4-D. Eight of the nine plasmids analyzed were identified as IncP-1ß plasmids with backbone sequences that are highly similar to those of the prototype 2,4-D-catabolic plasmid pJP4. These plasmids show minor differences in structure between each other indicating that they are variants of a similar ancestor. In one strain, the tfd genes were located on a plasmid, designated as pPO4, which appears to be a member of a poorly characterized plasmid group. The tfd genes on the IncP-1ß plasmids are highly similar in organization and sequence to the tfd genes identified on pJP4 although minor differences were detected. As on pJP4, the tfd genes are organized in two isofunctional catabolic clusters (tfd-I and tfd-II) that determine the conversion of 2,4-dichlorophenol into ß-ketoadipate and a separated tfdA gene that determines the conversion of 2,4-D to 2,4-dichlorophenol. Plasmid pPO4 carries a tfdA homologue and only one of the dichlorophenol catabolic gene modules. However, the strong similarity in sequence and organization of the tfd genes on pPO4 and the IncP-1ß plasmids suggest that the inter-plasmid exchange of tfd genes has taken place in the rice field bacterial community where the plasmids originated from. Moreover, the occurrence of the genes primarily on conjugative genetic elements with broad host range features shows the mobile character of the 2,4-D-catabolic phenotype in the examined soil community which can contribute to adaptation of its populations towards recruiting 2,4-D biodegradation genotypes. Novosphingobium sp. KN65.2 has the capability to use carbofuran as sole carbon and nitrogen source for growth that is accompanied by the mineralization of the aromatic ring structure of the compound. The genes involved in carbofuran degradation as well as the corresponding metabolic pathway in Novosphingobium sp. KN65.2 were analyzed. A collection of plasposon mutants impaired in carbofuran degradation and mineralization was isolated and representative mutants were subjected to proteomic and metabolite analyses. Metabolic profiling revealed several new metabolic intermediates, in addition to the initial hydrolysis product carbofuran phenol. The promiscuous carbofuran-hydrolyzing enzyme Mcd, which is present in several bacteria partially degrading carbofuran but lacking ring mineralization capacity, is not encoded by the KN65.2 genome. An alternative hydrolase gene required for this step was not yet 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 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, but appear also to be involved in carbofuran mineralization. The study has revealed the first dedicated carbofuran-catabolic genes and provides insight in the early steps of benzofuran ring degradation. The isolation of a carbofuran-mineralizing Novosphingobium sp. KN65.2 only from a carbofuran-treated field and the unique genetic make-up of carbofuran degradation genes in the strain supports the proposed hypothesis that continuously applied selection pressure of carbofuran in agricultural fields in the Mekong delta has resulted into a genetically adapted bacterial community that is able to degrade this intensively used insecticide.nrpages: 184status: publishe