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Appl Environ Microbiol, June 1998, p. 2051-2060, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Occurrence of a Sequence in Marine Cyanophages Similar to That of T4 g20 and Its Application to PCR-Based Detection and Quantification Techniquesdagger

Nicholas J. Fuller,1 William H. Wilson,2,* Ian R. Joint,3 and Nicholas H. Mann1

Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL,1 Marine Biological Association, The Laboratory, Plymouth PL1 2PB,2 and Plymouth Marine Laboratory, West Hoe, Plymouth, PL1 3DH,3 United Kingdom

Received 20 October 1997/Accepted 2 March 1998

Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity 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 region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100× concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately 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.

* Corresponding author. Mailing address: Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, United Kingdom. Phone: (44) 1752 633100. Fax: (44) 1752 633102. E-mail: whw{at}wpo.nerc.ac.uk.

dagger PRIME (Plankton Reactivity in the Marine Environment) contribution number 61.

Appl Environ Microbiol, June 1998, p. 2051-2060, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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