The molecular basis for the biotic degradation of metaldehyde John Christopher Thomas PhD University of York Biology September 2016 Abstract Metaldehyde is a molluscicide that is used to prevent crop damage by slugs and snails. It is frequently found in drinking water sources at concentrations above the legal maximum, and causes >90% of water quality failures in England. There is currently no economical, widely used treatment to remove metaldehyde pollution from water. Prior to the work presented here, it was known that metaldehyde is degraded biotically in soil, but the nature and identity of organisms responsible was unknown. Two novel metaldehyde degrading bacterial strains were isolated from domestic soil that can utilise metaldehyde as a sole carbon and energy source; Acinetobacter calcoaceticus E1 and Variovorax E3. Evidence that acetaldehyde is the primary metabolite of metaldehyde is presented: the metaldehyde dependent expression of an aldehyde dehydrogenase in A. calcoaceticus E1 was found, and the maximum metabolic flux of acetaldehyde was found to be greater than that of metaldehyde in cells grown using metaldehyde. The genomes of the isolated organisms were acquired. Comparative genomics of the two isolates showed that their novel catalytic ability was not dependent on genes shared between them, despite being isolated from the same soil sample. Comparison of the A. calcoaceticus E1 genome against other Acinetobacter that cannot utilise metaldehyde yielded a strong candidate for the primary metaldehyde degrading enzyme that has characteristics consistent with an enzyme that catalysed ether hydrolysis of isochorismate in its ancestral form and has evolved to hydrolyse ether bonds in metaldehyde. 2 Table of contents Abstract ................................................................................................................................................... 2 Table of contents ..................................................................................................................................... 3 Table of figures ........................................................................................................................................ 8 Table of tables ....................................................................................................................................... 12 Acknowledgements ............................................................................................................................... 14 Author’s declaration .............................................................................................................................. 15 Chapter 1: General Introduction ..................................................................................................... 17 1.1 The molecular diversity of soil microorganisms .................................................................... 17 1.2 The anthropogenic influence on soil ecology........................................................................ 18 1.3 Metaldehyde is a widely used molluscicide .......................................................................... 19 1.4 The toxicity of metaldehyde .................................................................................................. 20 1.5 Pollution of drinking water caused by metaldehyde ............................................................ 21 1.5.1 Metaldehyde use in other EU countries ........................................................................ 23 1.6 Chemical and physical methods of removing metaldehyde pollution .................................. 24 1.7 The biotic degradation of metaldehyde ................................................................................ 26 1.8 Bioremediation and biosensors ............................................................................................ 30 1.9 Aims of the project ................................................................................................................ 31 Chapter 2: Optimisation of a liquid-chromatography mass-spectrometry method for quantifying metaldehyde ....................................................................................................................................... 33 2.1 Introduction ........................................................................................................................... 33 2.2 Materials and methods ......................................................................................................... 35 2.2.1 LC and MS protocols ...................................................................................................... 35 2.3 Results ................................................................................................................................... 39 2.3.1 Decreasing response during, at least, the first 40 injections when analysing metaldehyde ................................................................................................................................. 39 3 2.3.2 No obvious sources of noise in parts of default protocol that take place before MS when analysing metaldehyde........................................................................................................ 40 2.3.3 The desolvation temperature significantly affects response and capillary voltage affects precision during the quantification of metaldehyde ......................................................... 51 2.3.4 Quantification of metaldehyde shows good precision across a range of concentrations but assessment of accuracy was weakened by standard preparations ........................................ 59 2.3.5 Comparison of precision of default protocol to optimised protocol ............................ 65 2.4 Discussion .............................................................................................................................. 66 2.4.1 Comparison to other published methods ..................................................................... 67 2.4.2 Summary ....................................................................................................................... 68 Chapter 3: Enrichment and characterisation of metaldehyde degrading organisms ..................... 69 3.1 Introduction ........................................................................................................................... 69 3.1.1 The isolation of microorganisms with specific metabolic capabilities .......................... 69 3.1.2 Bacterial phylogeny and the identification of species .................................................. 69 3.1.3 Aims ............................................................................................................................... 70 3.2 Materials and methods ......................................................................................................... 72 3.2.1 Minimal media ............................................................................................................... 72 3.2.2 Enrichments and isolations of metaldehyde degrading organisms .............................. 73 3.2.3 Substrate utilisation assays ........................................................................................... 74 3.2.4 Extraction of genomic DNA ........................................................................................... 75 3.2.5 16S polymerase chain reaction (PCR) and Sanger sequencing ..................................... 75 3.2.6 Genomic sequencing ..................................................................................................... 75 3.2.7 BLAST based method used to identify gnomically sequenced bacterial strains close to a strain isolated in this study ........................................................................................................... 76 3.2.8 Average nucleotide identity (ANI) ................................................................................. 76 3.3 Results ................................................................................................................................... 77 3.3.1 Acinetobacter strain E1 and Variovorax strain E3 were isolated in enrichments for metaldehyde catabolism ............................................................................................................... 77 4 3.3.2 Metaldehyde degrading organisms could not be isolated from agricultural soils or slow sand filter samples ........................................................................................................................ 81 3.3.3 The disappearance of metaldehyde from minimal media is proportional to the growth of Acinetobacter E1 and Variovorax E3 in pure cultures .............................................................. 82 3.3.4 Genomic sequence of isolates ....................................................................................... 86 3.3.5 Identification of A. calcoaceticus RUH2202 as a strain closely related to Acinetobacter E1 that does not degrade metaldehyde ........................................................................................ 90 3.3.6 Phylogeny of isolates E1 and E3 .................................................................................... 91 3.3.7 Carbon sources utilised by A. calcoaceticus E1 ............................................................. 94 3.4 Discussion .............................................................................................................................