The plant growth-promoting rhizobacterium Variovorax boronicumulans CGMCC 4969 was used to degrade the neonicotinoid insecticide, acetamiprid (AAP), in surface water, and the enzymatic mechanisms of AAP degradation in V. boronicumulans CGMCC 4969 were explored. V. boronicumulans CGMCC 4969 degraded 34.7% of 2 mg L−1 AAP over 120 h with a degradation half-life of 182 h in surface water, and the major metabolite was the amide product, (E)-N2-carbamoyl-N1-[(6-chloro-3-pridyl) methyl]-N1-methylacetamidine (IM-1-2). Gene cloning and over-expression studies proved that AAP hydration to IM-1-2 was mediated by a nitrile hydratase (ANHase). Addition of AAP to the mineral salt medium (MSM) broth significantly upregulated the ANHase gene expression by 1.6-fold, when compared with that in the control without AAP. Co-expression of the ANHase gene with its activator gene (anhC) apparently increased ANHase activity 21-fold for AAP hydration compared with the ANHase gene alone. The independent over-expression of anhC gave rise to competitive inhibition on the β-subunit of the ANHase and resulted in decreased ANHase activity. This ANHase is versatile, hydrating aromatic, N-heterocyclic, and aliphatic nitrile compounds. The present study shows the potential of V. boronicumulans CGMCC 4969 in the bioremediation of AAP contaminated water.

The nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333 and its nitrile hydratase (NHase) degrade the neonicotinoid insecticides, thiacloprid (THI) and acetamiprid (ACE), to their corresponding amide metabolites. The NHase gene cluster is composed of α-subunit and β-subunit genes and a hypothetical protein gene. The functionality of the hypothetical protein downstream of the NHase coding genes and the characteristics of CGMCC 7333 NHase were explored in this study. Co-expression of the hypothetical protein coding gene with NHase (α- and β-subunit genes) in Escherichia coli Rosetta enhanced NHase hydration of THI and ACE two- and four-fold, respectively, and also significantly improved NHase solubility compared with the absence of the hypothetical protein coding gene. The NHase displayed an optimal reaction temperature of 50 °C for THI hydration and was unstable when the incubation temperature exceeded 40 °C. The optimum reaction pH was 7.0 and the NHase activity was stable in the pH range of 6 to 9. The enzyme activity for THI hydration was slightly inhibited by copper, zinc, and iron, and decreased by 68.6%, 75.7%, and 70.3% when 2% ethanol, ethyl acetate, and acetone were added to the reaction mixture, respectively, whereas dichloromethane and trichloromethane had no effect. The Km and kcat values of CGMCC 7333 NHase for THI hydration were 12.39 mmol L−1 and 131.36 s−1, respectively. Substrate specificity analysis indicated that CGMCC 7333 NHase also transformed 3-cyanopyridine, benzonitrile, and indole-3-acetonitrile to the corresponding amide products, with maximum specific activities of 652.52, 255.32, and 263.93 U mg−1 protein, respectively.

Imidacloprid (IMI) is mainly metabolized via nitroreduction and hydroxylation pathways, which produce different metabolites that are toxic to mammals and insects. However, regulation of IMI metabolic flux between nitroreduction and hydroxylation pathways is still unclear. In this study, Pseudomonas putida was found to metabolize IMI to 5-hydroxy and nitroso IMI and was therefore used for investigating the regulation of IMI metabolic flux. The cell growth time, cosubstrate, dissolved oxygen concentration, and pH showed significant effect on IMI degradation and nitroso and 5-hydroxy IMI formation. Gene cloning and overexpression in Escherichia coli proved that P. putida KT2440 aldehyde oxidase mediated IMI nitroreduction to nitroso IMI, while cytochrome P450 monooxygenase (CYP) failed to improve IMI hydroxylation. Moreover, E. coli cells without CYP could hydroxylate IMI, demonstrating the role of a non-CYP enzyme in IMI hydroxylation. Thus, the present study helps to further understand the environmental fate of IMI and its underlying mechanism.

The metabolism of the widely used neonicotinoid insecticide acetamiprid (ACE) has been extensively studied in plants, animals, soils, and microbes. However, hydration of the N-cyanoimine group in ACE to the N-carbamoylimine derivate (IM-1-2) by purified microbes, the enzyme responsible for this biotransformation, and further degradation of IM-1-2 have not been studied. The present study used liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy to determine that the nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333 transforms ACE to IM-1-2. CGMCC 7333 cells degraded 65.1% of ACE in 96 h, with a half-life of 2.6 days. Escherichia coli Rosetta (DE3) overexpressing the nitrile hydratase (NHase) from CGMCC 7333 and purified NHase converted ACE to IM-1-2 with degradation ratios of 97.1% in 100 min and 93.9% in 120 min, respectively. Interestingly, IM-1-2 was not further degraded by CGMCC 7333, whereas it was spontaneously hydrolyzed at the N-carbamoylimine group to the derivate ACE-NH, which was further converted to the derivative ACE-NH2. Then, ACE-NH2 was cleaved to the major metabolite IM-1-4. IM-1-2 showed significantly lower insecticidal activity than ACE against the aphid Aphis craccivora Koch. The present findings will improve the understanding of the environmental fate of ACE and the corresponding enzymatic mechanisms of degradation.

Species of the genus Variovorax are often isolated from nitrile or amide-containing organic compound-contaminated soil. However, there have been few biological characterizations of Variovorax and their contaminant-degrading enzymes. Previously, we reported a new soil isolate, Variovorax boronicumulans CGMCC 4969, and its nitrile hydratase that transforms the neonicotinoid insecticide thiacloprid into an amide metabolite. In this study, we showed that CGMCC 4969 is able to degrade acrylamide, a neurotoxicant and carcinogen in animals, during cell growth in a mineral salt medium as well as in its resting state. Resting cells rapidly hydrolyzed 600 mg/L acrylamide to acrylic acid with a half-life of 2.5 min. In in vitro tests, CGMCC 4969 showed plant growth-promoting properties; it produced a siderophore, ammonia, hydrogen cyanide, and the phytohormone salicylic acid. Interestingly, in soil inoculated with this strain, 200 mg/L acrylamide was completely degraded in 4 days. Gene cloning and overexpression in the Escherichiacoli strain Rosetta (DE3) pLysS resulted in the production of an aliphatic amidase of 345 amino acids that hydrolyzed acrylamide into acrylic acid. The amidase contained a conserved catalytic triad, Glu59, Lys 134, and Cys166, and an “MRHGDISSS” amino acid sequence at the N-terminal region. Variovorax boronicumulans CGMCC 4969, which is able to use acrylamide for cell growth and rapidly degrade acrylamide in soil, shows promising plant growth-promoting properties. As such, it has the potential to be developed into an effective Bioaugmentation strategy to promote growth of field crops in acrylamide-contaminated soil.

A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis. Liquid chromatography–mass spectrometry and nuclear magnetic resonance analysis indicated the major pathway of thiacloprid (THI) metabolism by V. boronicumulans J1 involved hydrolysis of the N-cyanoimino group to form an N-carbamoylinino group containing metabolite, THI amide. Resting cells of V. boronicumulans J1 degraded 62.5% of the thiacloprid at a concentration of 200 mg/L in 60 h, and 98% of the reduced thiacloprid was converted to the final metabolite thiacloprid amide. A 2.6 kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an α-subunit of 213 amino acids and a β-subunit of 221 amino acids. The nitrile hydratase gene was recombined into plasmid pET28a and overexpressed in Escherichia coli BL21 (DE3). The resting cells of recombinant E. coli BL21 (DE3)-pET28a-NHase with overexpression of nitrile hydratase transformed thiacloprid to its amide metabolite, whereas resting cells of the control E. coli BL21 (DE3)-pET28a did not. Therefore, the major hydration pathway of thiacloprid is mediated by nitrile hydratase.