| dc.description.abstracteng | Bacterial viruses, known as bacteriophages or phages, are the most abundant biological entities on the planet and the least studied in terms of abundance and diversity. Searching the sequence databases of viral genomes, one becomes the impression that most of the viral sphere consists of dsDNA bacteriophages. First objective of the studies was to verify whether this is true or a methodical artefact of our usual approach of assessing the viral world. Second, bacterial host strains were needed for the investigation of bacteriophages. Third, besides the classic overlay plaque assay for isolation, the dsDNA, ssDNA, dsRNA, and ssRNA was also isolated from phage plaques as well.
To accomplish this endeavor, a local bacterial host system associated with various RNA and DNA viruses was required. Such hosts were not available at the beginning of this work and making it necessary to isolate a suitable prokaryotic system. To this end, environmental samples were successfully screened for new hosts, resulting in 37 new candidate bacterial strains, eight of which were sequenced and genomically analyzed (Brevundimonas pondensis, B. goettingensis, Serratia marcescens LVF3, Luteibacter flocculans, Stenotrophomonas indicatrix DAIF1, Kinneretia sp. DAIF2, and Janthinobacterium lividum EIF1 and EIF2). These were evaluated for their suitability as host systems (chapter 3.1 to 3.5, 3.8 and 3.9). A total of four new species were discovered and described. Using genomic analyses three of these were fully characterized (Brevundimonas pondensis, Brevundimonas goettingensis, and Luteibacter flocculans).
Brevundimonas pondensis LVF1 and Serratia marcescens LVF3 proved to be particularly promising candidates to achieve the main objectives of this thesis (chapter 3.7). They were used for classical phage isolation, resulting in 25 new dsDNA phages: 14 were associated with Brevundimonas and 11 with Serratia. TEM analysis revealed that six are myoviruses, 18 siphoviruses and one podovirus, while the Brevundimonas-associated phages are all siphoviruses. The classical approach was complemented by Next Generation Sequencing (NGS)-based methods that provided dsDNA, ssDNA, dsRNA, and ssRNA host-associated virome data. Furthermore, the complementary NGS approach enabled the identification of vB_SmaP-Kaonashi and vB_SmaM-Otaku. The latter is a virus that infects both host systems. In addition, the ssDNA virome associated with Brevundimonas pondensis revealed promising results as two contigs associated with ssDNA phages could be detected. These belong to the family Microviridae and Inoviridae. Further, the ssRNA virome of Brevundimonas goettingensis contained a contig associated with the Caulobacter-associated RNA phage phiCb5 which belong to the family Leviviridae.
In addition, bacteriophage isolates associated with the bacterial host strains Janthinobacterium (chapter 3.8) and Luteibacter (chapter 3.9) were found. Here, using the classical phage isolation approach, one phage was discovered for each bacterial host system. Janthinobacterium lividum produces the medically relevant antivacterial substance violacein. We were able to identify, that the induction of violacein is phage-dependent in this organism. Both phage isolates (Luteibacter phage vB_lflM-Pluto and Janthinobacterium phage vB_JliS-Donnerlittchen) are the first sequenced phages associated with the respective host systems.
In conclusion, the results of this work demeonstrates that dsDNA phages are the most prominent. Furthermore, the classical approach to phage isolation, which is still practical but biased, has successfully been demonstrated. Its limitations can be overcome by NGS-based methods to access viral diversity as efficiently as possible. | de |