Abstract
Marine cyanobacteria of the genera Synechococcus and Prochlorococcus are the most abundant photosynthetic organisms on earth, spanning vast regions of the oceans and contributing significantly to global primary production. Their viruses (cyanophages) greatly influence cyanobacterial ecology and evolution. Although many cyanophage genomes have been sequenced, insight into the functional role of cyanophage genes is limited by the lack of a cyanophage genetic engineering system. Here, we describe a simple, generalizable method for genetic engineering of cyanophages from multiple families, that we named REEP for REcombination, Enrichment and PCR screening. This method enables direct investigation of key cyanophage genes, and its simplicity makes it adaptable to other ecologically relevant host-virus systems. T7-like cyanophages often carry integrase genes and attachment sites, yet exhibit lytic infection dynamics. Here, using REEP, we investigated their ability to integrate and maintain a lysogenic life cycle. We found that these cyanophages integrate into the host genome and that the integrase and attachment site are required for integration. However, stable lysogens did not form. The frequency of integration was found to be low in both lab cultures and the oceans. These findings suggest that T7-like cyanophage integration is transient and is not part of a classical lysogenic cycle.
Highlights
Marine cyanobacteria of the genera Synechococcus and Prochlorococcus are abundant primary producers that span vast areas of the oceans [1]
Cyanophage genomes contain many intriguing and unusual features. They encode a variety of “auxiliary metabolic genes” (AMGs) [10, 14, 22,23,24,25] ofbacterial origin, including those linked to photosynthesis [26, 27], carbon metabolism [28] and nutrient utilization [18, 29], as determined from homology-derived functional assignments
A large reservoir of diverse genes with no putative function is present in cyanophage genomes [16,17,18, 22], as found for other viruses in the environment [30]
Summary
Marine cyanobacteria of the genera Synechococcus and Prochlorococcus are abundant primary producers that span vast areas of the oceans [1] Their viruses (cyanophages) are highly abundant [2,3,4] and influence cyanobacterial ecology and evolution [5,6,7], as well as global biogeochemical cycles [8,9,10]. The T4-like cyanophages from the Myoviridae family and the T7-like cyanophages from the Podoviridae family share sets of core genes with the Escherichia coli T4 and T7 phages, respectively [13, 15, 18, 22] These genes are involved in virion formation, DNA replication and packaging, transcription and other fundamental processes of the lytic infection cycle. Some of these genes are conserved in multiple cyanophage types [16, 18, 21, 22] whereas others have no database match, and are termed “ORFans” [30, 31]
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