OCCURRENCE OF A SEQUENCE IN MARINE CYANOPHAGES SIMILAR TO THAT OF T4 G20 AND ITS APPLICATION TO PCR-BASED DETECTION AND QUANTIFICATION TECHNIQUES

Viruses are ubiquitous components of marine ecosystems and are known t o infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage gen ome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant si milarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2 which specifically amplified a 165-bp regi on from the majority of cyanomyoviruses tested. A competitive PCR (cPC R) analysis revealed that cyanomyovirus strains could be accurately en umerated, and it was demonstrated that quantification was log-linear o ver ca. 3 orders of magnitude. Different calibration curves were obtai ned for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial in accuracies in estimates of phage abundance in natural assemblages, Fur ther sequence analysis of cyanomyovirus gene 20 homologs would be nece ssary in order to design primers which do not exhibit phage-to-phage v ariability in priming efficiency. It was demonstrated that PCR product s of the correct size could be amplified from seawater samples followi ng 100x concentration and even directly without any prior concentratio n. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophag es in natural assemblages. Further optimization of procedures may ulti mately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products.