Molecular approaches to the study of marine cyanophages
Cyanophages are thought to play an important role in the mortality and clonal composition of marine Synechococcus spp., and have been shown to be widespread throughout the world's oceans. However, relatively little research has been made into the molecular analysis of marine cyanophages. This study continued previous research to develop molecular probes (PCR primers) which would specifically detect cyanophages which infect marine Synechococcus spp., and be used to interrogate natural marine cyanophage populations. An attempt was made to develop a rapid technique for quantifying marine cyanophages, using competitive PCR (cPCR). For the development of cyanophage-specific PCR primers, several cyanophages which infected Synechococcus sp. strains WH7803 and WH80 18 were isolated from coastal Bermuda and the Sargasso Sea. A region of DNA had previously been found which showed homology amongst several marine cyanophages, and to T4 gene 20, which encodes a minor capsid protein. Homologues from three cyanophages were completely sequenced, and two, potentially cyanophage-specific, PCR primers were designed. The primers detected only marine cyanophages which belonged to the family Myoviridae, regardless of the geographical location of their isolation. They also detected cyanophages which infected different marine Synechococcus spp. strains, and therefore provide a more comprehensive tool than infective methods. The primers were able to detect as few as 190 cyanophages Ilr1, which would correspond to an in situ concentration of 103 PFU mH. The PCR should therefore detect most natural concentrations of marine cyanophages in surface waters, especially with prior concentration from seawater. Preliminary experiments showed that PCR products could be obtained from as little as I III of un concentrated seawater. PCR therefore provides a sensitive method for the detection of marine cyanophages, which is far more rapid than traditional infection techniques. Quantification by cPCR was attempted. An internal competitor was constructed, and a calibration curve was drawn for three cyanophages, with a loglinear relationship over ca. three orders of magnitude of cyanophage numbers. This demonstrates that rapid quantification of a known marine cyanophage is possible. However, cPCR of the three different cyanophages resulted in three different calibration curves. Hence, quantification of a marine sample containing a mixture of cyanophages was not yet possible. The cyanophage-specific primers were then applied to marine samples which were collected whilst on the AMT-2 cruise, from Port Stanley (Falkland Islands) to Plymouth (UK). Cyanophages were concentrated by tangential flow filtration, and PCR products were obtained from most of the surface samples throughout the Atlantic Ocean. Products from some of the stations were sequenced, providing novel genetic information of natural marine cyanophage populations. The results showed that cyanophage populations were highly diverse, with at least twelve genetically different cyanomyoviruses in one sample. Some sequences obtained from the same sample were clearly very similar to each other, whilst others within a sample could be as diverse as those isolated from different oceans. However, very similar sequences were obtained from some samples separated by thousands of miles, in different hemispheres, or even in different oceans.