Cosubstrates Regulate The Metabolic Pathway Of Neonicotinoid Insecticide Imidacloprid And By Stenotrophomonas Maltophilia Strain R551-3

The metabolism of neonicotinoid insecticide imidacloprid (IMI) has been extensively studied in soils, plants and mammals; however, the regulation of IMI metabolic pathway is still poorly understood. In the present study, we compared the regulation of IMI hydroxylation and the hydroxylated metabolite's dehydration as well as IMI nitroreduction by a genome well-known bacterium, Stenotrophomonas maltophilia R551-3, in response to sucrose and succinate as cosubstrates.In order to analyzing the metabolic pathways and metabolites of IMI quantitatively, the substituents of IMI (=N-NO2) such as guanidine IMI (=NH), nitroso IMI (=N-NO), urea IMI (=O) and olefin IMI were synthesized chemically and 5-hydroxy IMI was synthesized by microbial tansformation. The substituents of IMI were then used for quantitative HPLC analysis.In the presence of sucrose as a cosubstrate S. maltophilia R551-3 transforms IMI to form the major metabolite 5-hydroxy IMI and a few spontaneous hydrolysis product olefin IMI. Compared with the control, the hydroxylation is enhanced eight times upon the addition of sucrose, while the nitroreduction pathway is completely inhibited. In contrast, both the hydroxylation and nitroreduction pathways were observed in the presence of succinate as a cosubstrate with the metabolites of 5-hydroxy, olefin, urea IMI and an unknown product (retention time 3.1 min). In the presence of sucrose and succinate as cosubstrates,23.1% and 57.6% IMI were degradated respectively at the eighth day. So succinate can more facilitate the degradation of IMI than sucrose. 5-hydroxy IMI is cometabolically converted to olefin IMI in the presence of succinate, while not in the presence of sucrose. Nitroso IMI is converted to urea and guanidine IMI in the presence of succinate, while not in the presence of sucrose. Neither guanidine nor urea IMI could be transformed in the presence of succinate or sucrose, which indicates that guanidine IMI is not converted to the end-product urea IMI by S. maltophilia R551-3. 61.8% of reduced IMI and 60.0% of 5-hydroxy IMI formation were inhibited by the inhibitors of the hexosemonophosphate pathway (HMP) 6-aminonicotinamide which indicates that the HMP is the main metabolic pathway of sucrose in hydroxylation of IMI by S. maltophilia R551-3. Pyruvate and malate facilitate olefin IMI formation and do not inhibit urea IMI formation. Additional acetate partially terminates the inhibition of IMI nitroreduction to urea IMI in the presence of sucrose.22.2% of urea IMI formation can be inhibited by NADPH, whlile can't by NADH. The above results indicate that sucrose metabolized by HMP will generate NADPH which can inhibit IMI nitroreduction pathways. Therefore the mechanism of the metabolic pathway of IMI regulated by different cosubustrates was controled by the intracellular metabolic flow of cosubstrates. NADPH is generated when the co substrate is metabolized by HMP pathway, which can facilitate IMI hydroxylation and inhibit IMI nitroreduction and 5-hydroxy IMI dehydration to olefin IMI. IMI nitroreduction and 5-hydroxy IMI dehydration to olefin IMI are not inhibited when the co substrate is metabolized by EMP pathway or TCA cycle.The differences of IMI cometabolism between S. maltophilia R551-3 and CGMCC 1.1788 were further compared. In the presence of succinate as cosubstrate, no apparent differences were observed between IMI nitroreduction (urea IMI formation) by the resting cells of S.maltophilia R551-3 and CGMCC 1.1788. While the reduced IMI by the resting cells of S. maltophilia CGMCC 1.1788 was nearly twice fold higher than strain R551-3, and the formation of olefin IMI by the resting cells of S. maltophilia CGMCC 1.1788 was 8.3 times more than R551-3; Olefin IMI was not generated by the cell-free extract of S. maltophilia CGMCC 1.1788, while was by R551-3. In the presence of sucrose as cosubstrate, the IMI hydroxylation by the resting cells of S. maltophilia CGMCC 1.1788 was 5.6 times more than that by S. maltophilia R551-3. The IMI hydroxylation by the cell-free extract of S. maltophilia CGMCC 1.1788 declined sharply, while the IMI hydroxylation by the cell-free extract of S. maltophilia R551-3 was almost equal to intact cells. In the presence of succinate as the carbon sources, the reduced IMI by the growing cells of S. maltophilia CGMCC 1.1788 was higher than R551-3. In the presence of sucrose as the carbon sources, the IMI hydroxylation by the growing cells of S. maltophilia CGMCC 1.1788 was higher than R551-3. The study showed that the differences of IMI cometabolism between S.maltophilia R551-3 and CGMCC 1.1788 were remarkable.