Tue Sparholt Jørgensen:
Characterization of the mobilome and microbiome of rat cecum

Date: 15-04-2015    Supervisor: Søren J. Sørensen & Lars Hestbjerg Hansen

Where eukaryotic recombination relies on sex between two individuals of the same species, prokaryotic recombination is much, much more promiscuous. Here, an almost universal genetic language allow for traits to be shared between existing cells of different Family, Phylum and even Kingdom. Extrachromosomal genetic elements are the vectors of this recombination and are thus pivotal in bacterial evolution yet little research has focused on elements outside of laboratory culturing. The shapes of extrachromosomal elements are many, with both linear and circular variants, varying in size from few hundred bases to millions of bases and with and without a protein encapsulated extracellular state. In this thesis, I have focused on circular extrachromosomal elements and as a first automated identification of complete elements, yielding thousands of circular sequences.

The thesis is divided in two chapters, an introductory chapter where the biological background and methods of the thesis is explained, with a focus on subjects and opinions not covered in the introductions in the manuscripts chapter two. This thesis has resulted in two publications and two manuscripts, along with a number of collaboration, of which hopefully some will lead to publication and others to further collaborations.

This thesis is based on the biological material squeezed out of the cecum of some 30 rats collected from the following five environments: Falkland Islands, North Jutland, two hospital sewer sites in Copenhagen and an animal experimental laboratory. 16S rDNA amplicons, mobilomes and metagenomes were produced from these samples in order for the different data types to shed light on different aspects of microbial life in the rat gut. Integration of these data types is still not fully utilized, with a number of factors limiting its application.

In Manuscript 1 “Hundreds of Circular Novel Plasmids and DNA Elements Identified in a Rat Cecum Metamobilome”, the main concepts and methods of the mobilome part of this thesis are developed, particularly the pipeline that allow identification of complete circular extrachromosomal units. 616 circular elements were identified from a single sample. Of these 614 were novel and are deposited in the NCBI database, substantially adding to the known circular extrachromosomal units. An important prerequisite was the identification and test of the effect of interspersed repeats and circularity on assembly, allowing a separation of the two. Further, calculations of the chromosomal DNA content revealed that only few percent stem from this pool, why the potentially problematic long repeat issue is not relevant for mobilome samples. Finally, many novel plasmid replication proteins were identified, very distantly related to any known nucleotide sequence. In the perspectives Manuscript 2 ”Current strategies for mobilome research”, the approaches of different research groups to examining entire extrachromosomal landscapes is reviewed and compared and possible future directions of research are considered. Manuscript 3 TITLE HERE applies the methods developed in Manuscript 1 on 12 deeply sequences mobilome samples. Further methods are developed to separate and categorize the identified 1,869 circular DNA sequences identifying more than 500 plasmids, more than 100 viruses and more than 1,000 uncategorized elements. Interestingly, ca 10% of the circular sequences are found in multiple samples and some even in Falkland Island samples and Copenhagen sewer samples, highlighting that the elements are not limited to a single geographical or environmental biotope.

In Manuscript xx, microbiome of Rattus norvegicus the diversity of bacteria of wild and laboratory brown rats are explored on the basis of 16S rDNA amplicon sequencing. The DNA precursor of the small 16S subunit of ribosomal RNA has become the standard in microbial classification and is here used to determine a bacterial flora dominated by Firmicutes and Bacteroidetes, a pattern also seen in other hind gut fermenters. A tendency of animals expected to ingest a lot of dietary fiber to have increased numbers of Firmicutes Clostridia was observed and notably, different dominating taxa was present in the laboratory rats than in the wild rats. Further, a clear separation of environments based on OUT presence and abundance suggest that environmental factors supersede the individual variation of microbiomes.