Peter Nikolai Holmsgaard:
Second generation sequencing for elucidating the diversity of bacteria and plasmids in soil

Date: 15-08-2013    Supervisor: Søren J. Sørensen & Lars H. Hansen

Soil is considered to one of the most diverse microbial environments on Earth and the study of this diversity has in recent years been revolutionized by amplicon pyrosequencing of the 16S rRNA gene. The focus of this thesis is the use of amplicon pyrosequencing to study not only bacterial diversity but also plasmids diversity in soil. The plasmid group in focus are the broad host range IncP-1 plasmids that were studied by amplicon pyroseqeuncing of the trfA gene encoding the replication initiation proteins. The thesis consists of an introduction spanning microbial ecology, IncP-1 plasmids and amplicon pyrosequencing and briefly the related bioinformatics. This is followed by six papers of which one is published and five are manuscripts.

In the first paper, amplicon pyrosequencing of the 16S rRNA gene was use to investigate the bias in cell extraction from soil imposed by Nycodenz density gradient centrifugation. The second paper proves the possibility of simultaneous amplicon pyrosequencing of the 16S rRNA gene, trfA gene and merA gene. In addition, it was determined that the IncP-1 subgroups can be distinguished by the trfA amplicons at 89% nucleotide sequence similarity.

In the third, paper microcosms with material from a pre-adapted biopurification system (BPS) were spiked with the herbicide linuron and the effect on the bacterial and plasmid community was studied. The genus Variovorax previously shown to degrade linuron was found to be one of the main positive responders. The relative abundance of IncP-1β1 plasmids also increased. In papers four and five, the mobile genetic elements and bacterial diversity, respectively, was studied over a pesticide spraying season in the same BPS used in paper three. The addition of pesticides decreased overall bacterial diversity and increased the relative abundance of Gammaproteobacteria. Additionally, a high abundance of IncP-1, IncP-7, IncP-9, IncQ and IncW plasmids and class I and II integrons were detected. Amplicon pyrosequencing of the IncP-1 plasmids showed an increase in relative abundance of the IncP-1β and decrease of IncP-1ε during the season.

In the sixth paper, unspecific DNA amplification using multiple displacement amplification was shown to preferentially amplify DNA from some bacterial species over others.

In conclusion, papers II to V use, among other things, amplicon pyrosequencing to elucidate the bacterial and plasmid diversity in different soil environments while papers I and VI use different molecular methods to study the bias imposed on bacterial community studies by different techniques.