Identification and characterization of the biological mechanisms of metaldehyde degradation

Metaldehyde is a molluscicide used to protect agriculture, horticulture and domestic crops from slugs and snails. Following its application, metaldehyde has been demonstrated to wash-off into nearby water sources and as such has been frequently detected above the EU statutory drinking water limit of 0.1 µg/L. Due to metaldehyde’s chemical properties, current water treatment processes are currently economically unsustainable. Prior to the work conducted within this thesis, several metaldehyde degrading bacteria had been isolated and identified. However, the degradative enzymes responsible were unknown. The work provided here involved the discovery and experimental verification of the first metaldehyde degrading enzymes. Random chemical mutagenesis of metaldehyde-degrading strain Acinetobacter calcoaceticus E1 using the mutagen ethylmethanesulphonate led to the isolation of four mutants deficient in metaldehyde degradation. Comparative genomic analysis of the mutants resulted in the discovery of a catabolic gene cluster hypothesized to be responsible for metaldehyde degradation. This cluster contained a predicted Fe (II)/ (alpha) ketoglutarate-dependent dioxygenases (MahX), a lyase (MahY) and an aldehyde dehydrogenase (MahZ). Heterologous expression within Escherichia coli revealed the role of MahX as the initial degradative enzyme for catalysing metaldehyde. Based on the enzymes identified within this research, a predicted metaldehyde degradation pathway was proposed. Bioinformatic analysis based on the MahX protein sequence allowed for the identification and experimental verification of a novel metaldehyde degrading protein MahS within the metaldehyde degrading strain Sphingobium sp CMET-H. Genomic analysis revealed mahS to be located within a 194 kbp conjugative plasmid suggesting the mobile nature of the degradative gene. Protein characterization of MahX and MahS demonstrated the catabolic activity of the enzymes in vitro and revealed the requirements for the cofactors Fe2+, alpha-ketoglutarate and a reducing agent to achieve optimum degradation. This research has expanded the knowledge regarding the biological degradation of metaldehyde and provides the basis for targeted bioremediation and biomonitoring approaches.