ISOLATION, GENOMICS AND ECOLOGY OF BACTERIOPHAGES INFECTING MARINE ROSEOBACTERS
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Viruses are the most abundant biological entities in seawater. They influence the population dynamics, genetic heterogeneity, and biogeochemical cycles in marine ecosystems. Isolation and characterization of viruses which infect specific hosts have greatly advanced our knowledge on the biological and ecological interactions between viruses and their hosts. Roseobacter is an important lineage of marine bacteria which are genetically diverse, abundant and ubiquitous in the ocean. Roseobacters can make up to 25% of bacterial communities in coastal environments and play an active role in the marine sulfur cycle. However, only few bacteriophages which infect marine roseobacters had been isolated at the time when I began my studies. To understand the types of bacteriophages that infect roseobacters and how they interact with their hosts, I devoted my research to isolation and characterization of the bacteriophages infecting roseobacters (roseophages hereafter). In this dissertation, fourteen different phages infecting a marine strain, Ruegeria pomeroyi DSS-3, are described in terms of their morphology, growth, genomics and global distributions. These 14 roseophages were divided into four different groups: ssDNA, CbK-like, Chi-like, and N4-like roseophages. Two ssDNA phages belongs to an unclassified group of Microviridae. They contain only four ORFs with a genome size of 4.2 kb, representing the smallest and of all known ssDNA phage isolates. Interestingly, the ssDNA roseophages fall into a large group of uncultivated viral sequences identified by viral metagenomics. The isolation of CbK-like roseophages uncovers a new type of Siphoviridae infecting a member of Roseobacter lineage, Prior to this work, CbK-like phages had only been reported in a freshwater bacterium Caulobacter. The two CbK-like roseophage genomes are highly mosaic, containing features from siphoviruses, podoviruses, gene transfer agents, integrases and a large number of tRNAs. Chi-like siphophages are another newly discovered group of roseophages. Five different Chi-like phages (Siphoviridae) were isolated from DSS-3. A resistant strain of R. pomeroyi DSS-3 was found during superinfection with Chi-like roseophage DSS3Φ1. Genome sequencing confirmed that the resistant strain contains the intact genome of DSS3Φ1. The ability to integrate phage genome into host chromosome confirms that DSS3Φ1 is a temperate phage. Five N4-like roseophages of DSS-3 were isolated. They belong to the phage N4 lineage in Podoviridae. Genomes of N4-roseophages are highly syntenic, sharing a very similar genomic arrangement. The genomic conservation of N4-like phages allowed me to design N4-like phage specific primers based on their DNA polymerase genes. The primer set was used to PCR amplify the DNA pol gene of N4-like phages from 56 DNA samples to investigate the diversity and distribution of N4-like phages in the Chesapeake Bay. Surprisingly, N4-like phage sequences were only detected in the winter samples collected over two years. Metagenomic recruitments also confirmed that N4-like phages appear to prevail in the cold environment, such as Organic Lake, a hypersaline lake in Antarctica, where the temperature is usually below -10 C. According to metagenomic analyses, homologs of other DSS-3 phages (non-N4-like) are present in freshwater and marine habitats, Antarctica, human gut and feces, and coral-associated environments. This wide range distribution of roseophages seems to reflect the cosmopolitan nature of the Roseobacter clade. The discovery of different types of phages infecting a single strain and their wide distribution suggest that we are only seeing the tip of the iceberg of phages.