Acquisition and ongoing evolution of synthetic xenobiotic metabolic pathways in soil bacteria
Creators: name: Pearce, Stephen Leslie
Publisher: name: The Australian National University
Year: 2015

description: Man-made toxic chemicals have the potential to cause serious harm to humans and ecosystems. The long half-lives of many of these chemicals mean that they will remain a problem well into the future and require intervention to limit their harmful effects. One important approach to deal with these compounds is microbial bioremediation, which involves the use of bacterial strains with metabolic pathways capable of transforming these compounds into non-toxic products. These pathways provide an excellent model for studying the evolution of new metabolic pathways as they have all developed very recently in evolutionary time. This study explores the evolution of two metabolic pathways, one that is in the very early stages of evolution and one that has undergone secondary changes after being initially acquired. The first pathway looks at the metabolism of 6-chloronicotinic acid, an important intermediate in the breakdown of several widely used neonicotinoid insecticides. The isolation of a 6-chloronicotinic acid mineralising bacterium (strain SG-6C) is described and a novel metal-dependent chlorohydrolase (cch2) is identified that has potential applications in the bioremediation of neonicotinoids. Further, cch2 is revealed to have been acquired through horizontal transfer of an Integrative and Conjugative Element, which remains an active mobile element in SG-6C. The additional steps in mineralisation are shown to be provided by a pre-existing nicotinic acid degradation pathway common to many soil dwelling proteobacteria. This suggests that there is a large pool of strains with the potential to become 6-chloronicotinic acid mineralising strains through horizontal transfer of cch2. The second pathway to be examined is the lin pathway, for metabolism of the organochloride insecticide lindane. The complete lin pathway is known and, like the 6-chloronicotinic acid pathway, has a horizontally transferred upstream and a pre-existing downstream component. A comparative analysis of the genomes of ten lindane metabolising strains (including three sequenced in this study) shows the dynamic nature of the pathway, with several instances of duplicated or missing genes. The important role of insertion sequence IS6100-mediated horizontal transfer in the acquisition of the pathway is also confirmed. IS6100 elements are shown to be the key factor in widespread transfer of the upstream pathway and are found associated with the downstream lin genes for the first time. These evolutionary dynamics have a number of implications for the design of lindane bioremediation strategies. Finally, the genome sequencing performed for this project identified an uncharacterised variant of linA, encoding a dehydrochlorinase responsible for the initial steps of lindane metabolism. This variant is shown to have a novel degradation phenotype, exhibiting an altered isomer preference compared to all other known LinA enzymes. A computational analysis of this variant suggests that two residue differences in particular are responsible for this changed phenotype. Analysis of the sequence of this LinA reveals it is a representative of a new class of LinA variants (LinA-type3) that have proliferated rapidly and recently due to positive selection. These rapid changes suggest that the lin pathway will continue to provide important evolutionary insights as it matures.