Packaging of Genetic Material by Gene Transfer Agents (GTAs) Produced by Marine \u3cem\u3eRoseobacter\u3c/em\u3e Species and Their Effect on Stimulating Bacterial Growth

Horizontal gene transfer is one of the most important mechanisms for prokaryotic genome innovation and evolution. Gene Transfer Agents (GTAs) are phage-like particles that package small fragments of the genome of a GTA-producing bacterial cell. GTA chromosomal gene clusters usually contain 15-conserved open reading frames (ORFs) and are present in most of the sequenced marine alpha-proteobacteria genomes. Some marine strains have been shown to produce GTA particles that were biologically active in marine environment. GTA particles range in size, morphology and the amount of host DNA they package. To date, the characteristics of GTAs are largely based on observations of Rhodobacter capsulatus, a bacterial isolate from freshwater pond and soil samples. One of the main characteristics of the GTAs produced by R. capsulatus is random packaging of the genetic contents of the GTA-producing strain. However, there is no evidence that marine GTAs behave in a similar manner to those produced by R. capsulatus. This thesis focuses specifically on the GTAs produced by marine bacterial isolates, aiming to expand the available knowledge of how GTAs of marine bacterial strains contribute to HGT and how they affect the bacterial adaptation and fitness in the ocean. Here, the putative GTA particles produced by marine bacterial strains grown in artificial seawater media were examined to investigate the randomness of the DNA packaging and the biological effect of the GTA particles, specifically examines the effect of GTAs on stimulation of bacterial growth in vitro. To reach the desired outcome, first, the DNA packaged within GTA particles produced by Roseovarius nubinhibens (RnGTA), Ruegeria pomeroyi (RpGTA) and Roseobacter denitrificans (RdGTA) was sequenced to determine if random portions of the bacterial genomes are packaged, similar to results shown for R. capsulatus, or if certain areas of the genome are preferentially packaged (overrepresented). Further, purified active GTA particles derived from each of the three marine Roseobacter strains were tested to determine the effect of active GTA particles on bacterial growth compared to controls containing heat-inactivated GTA particles, induced prophages (where applicable) and buffer. In summary, the production of GTA particles produced by R. denitrificans was observed for the first time. Additionally, the results of sequencing, annotating and assembling the packaged DNA within GTAs from the marine Roseobacter strains that were studied here suggested that although there was a good representation of the whole genome packaged within the GTA particles, still there is significant enrichment (overrepresentation) of gene groups that could expand their metabolic capabilities. Also, in vitro, under nutrient replete conditions, GTA particles of R. nubinhibens (RnGTA) seemed restricted to having impact on growth to members of the same species. On the contrary, when seawater samples were treated with GTAs there was increase in viable cell counts. By closely examining the colony morphologies, there was a clear difference between the bacterial species that grew when seawater samples were treated with RpGTA and RdGTA compared to controls. The 16S rRNA identifications revealed that under the tested laboratory settings, some species belonging to phylum Flavobacterales are more responsive to active GTA treatment than others, causing microbial community shift in seawater samples. This study has expanded what is known about GTAs of marine origin, providing genetic and metabolic evidence that GTAs may stimulate microbial diversity and survival in the marine environment. Knowledge gained from this study will help us understand the role of GTAs and HGT mechanisms in the ocean, therefore advancing our knowledge about the evolution and interaction of marine microbes