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The genetic analysis of aquatic cyanophage communities Frederickson, Cindy Marie

Abstract

Viruses are abundant in the marine environment and are important in terms of carbon and nutrient cycling. Through the specific lysis of host organisms, they play a role in structuring host communities of phytoplankton and bacteria. Phage, viruses that infect bacteria, are the most abundant virus-like particles in seawater, but relatively little is known about their biology, phylogeny and diversity. Previously, sequence analysis revealed a region of similarity among the genomes of three cyanophages belonging to the family Myoviridae and the capsid assembly gene (g20) of coliphage T4. These sequences were used to design two sets of putative cyanophagespecific PCR primers (CPS4GC/CPS5 and CPS4GC/CPS9). In the first part of the study, natural viral communities were collected from 6 depths in each of 3 inlets in British Columbia, Canada. Cyanophage-specific primers CPS4GC and CPS 5 were used to amplify -205 bp (including 40 bp GC-clamp) g20 gene fragments from the samples, and denaturing gradient gel electrophoresis (DGGE) was used to fingerprint the natural cyanophage communities. The results showed that the cyanophage communities differed the most within inlets in which temperature and salinity changed the most with depth. Differences in cyanophage communities sometimes occurred in association with high chlorophyll fluorescence and/or high abundances of infectious cyanophage and Synechococcus cells. In order to obtain more sequence for phylogenetic analysis, a new primer (CPS9) was designed and used with CPS4GC to amplify a 595 base pair region of the g20 gene. These primers were used to amplify gene fragments from lakes in British Columbia and Germany, a catfish production pond in Louisiana, a cyanobacterial mat from a high arctic pond, as well as samples from both polar oceans, the Gulf of Mexico, northeast Pacific inlets and the coastal southeast Pacific. DGGE was used to separate the fragments, which were subsequently excised, re-amplified, cloned and sequenced. Phylogenetic analysis demonstrated high variability in the g20 gene sequences and revealed four previously unknown groups of phage, two of which consisted of sequences found only in freshwater. Sequences that were >99% identical in terms of pairwise nucleotide similarity were recovered from both freshwater and marine environments, suggesting that genetically similar phage are globally distributed. My research has demonstrated that PCR combined with denaturing gradient gel electrophoresis can be used to compare marine cyanophage communities. I have shown that these communities can differ on spatial scales as small as metres and that this is influenced by the physical structure of the water column. As well, by PCR amplifying the g20 gene from geographically distinct environments, I have shown that this group of phage is global in distribution. I have demonstrated the genetic richness of this group and have potentially identified new groups of freshwater phage.

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