Developing a Sampling Strategy for Antibiotic Bioprospecting from Bacteria in Terrestrial Ecosystems.

Abstract

This thesis investigates the problem of how to sample terrestrial environments for bioprospecting for pharmaceutically useful bacterial secondary metabolites. There is already a considerable literature on the ecology of bacteria in terrestrial ecosystems as well as on attempts to bioprospect from particular terrestrial environments. The first chapter reviews both of these kinds of studies and outlines unresolved questions that need to be answered to efficiently sample for bioprospecting. Most studies of bacterial communities in soil use molecular methods and start by extracting DNA by bead beating. The extracted DNA is then PCR amplified using 16S bacterial primers. Bead beating is usually done with the Mobio kit, however, there is no one standard 16S primer pair that is used in most studies. The second chapter tests how comparible results from these studies are. DNA was extracted from soil and compost using the Mobio kit and a contrasting method and amplified with four different primer pairs. Results showed that primers have more effect on results than extraction method but that the Mobio kit underextracts several genera by orders of magnitude. Most antibiotics from bacteria that are in use come from the Actinobacterial phylum. The third chapter uses several primer pairs for Sanger sequencing and bacterial community fingerprinting to compare the Actinobacterial community structure of a range of environments under different Anthropogenic impact. It found that street sediments were enriched in Actinomycetes compared to soil. In the fourth chapter, environmental DNA from soil and street sediments was amplified with primers for type I polyketide synthase (PKSI) genes. Amplicons were sequenced with Sanger sequencing. Several PKSI amplicons were common to soil or street sediment from 1000s of km apart. Soil PKSI sequences were more often non Actinomycetal and more novel than street PKSI sequences.