Dissertation Defense Seminar: Yuanchao Zhan, UMCES-IMET

Title: ISOLATION, GENOMICS AND ECOLOGY OF BACTERIOPHAGES INFECTING MARINE ROSEOBACTERS

Speaker: Yuanchao Zhan, Ph.D. Candidate, UMCES-IMET

Abstract: 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 hosts. Roseobacter is an important lineage of marine bacteria which are abundant, ubiquitous and diverse in the ocean. Roseobacters can make up 25% of bacterial community in the coastal water and play an active role with sulfur cycle in the marine environment. However, only few bacteriophages which infect marine roseobacters have been isolated at the time when I started my Ph.D study. To understand what kind of bacteriophages infect roseobacters and how they interact with roseobacters, I devoted to isolate and characterize the bacteriophages infecting roseobacters (roseophages hereafter).

                In this dissertation, fourteen different phages infecting a marine roseobacterial strain, Ruegeria pomeroyi DSS-3 are described in terms of their morphology, growth, genomics and global distribution. These 14 roseophages were divided into 4 different groups: ssDNA, CbK-like, Chi-like, and N4-like roseophages. Two ssDNA phages which belong to an unclassified group of Microviridae were first found to infect marine roseobacters. They also represent the first isolation of ssDNA phages infecting the major phylum of bacteria - Proteobacteria. The ssDNA roseophages only contain four ORFs with a genome size of 4.2 kb, representing the smallest and simplest ssDNA phages among all the known ssDNA phage isolates. Interestingly, the ssDNA roseophages fall into a large group of unknown viral sequences uncovered by viral metagenomics. The isolation of CbK-like roseophages uncovers a new type of phage (Siphoviridae) infecting a member of Roseobacter lineage, and this type of phage has not been reported in marine bacteria. CbK-like phages were only reported in a freshwater bacterium Caulobacter prior to this study. The two CbK-like roseophages 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 the 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 us to design the 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 2 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 the metagenomic analysis, 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 roseobacterial stain and their wide distribution suggest that we are only seeing the tip of iceberg of roseobacterial phages.