Marine Synechococcus strains are abundant and ecologically important cyanobacteria, yet their genetic manipulation remains technically challenging due to their polyploidy, limited DNA uptake mechanisms, and low transformation efficiency. Cyanophages are viruses that specifically infect marine cyanobacteria. In this study, we aimed to develop a reliable genetic toolkit for Synechococcus sp. WH7803 to enable functional genomics and cyanophage interaction studies. First, we optimized an electroporation-based protocol for introducing a suicide plasmid into WH7803. Using a knockout construct targeting the SynWH7803_1017 locus, we demonstrated successful plasmid delivery and homologous recombination via electroporation at 1.5 kV, with PCR validation confirming gene disruption.
Building on this platform, we next investigated whether large cyanophage genomes could be introduced into WH7803 via electroporation. Electroporation of purified phage Syn9 DNA induced host physiological changes, such as fluorescence decline and pigment loss, resembling those seen during natural infection, although no viral replication or lytic release was detected. These observations suggest that electroporated phage DNA may be internalized and exert limited functional effects on the host, even in the absence of productive infection.
Finally, to identify host genes involved in phage resistance, we focused on a spontaneous mutant (WH7803-4a3) harboring a mutation in SynWH7803_0948, a putative multicopper oxidase. We constructed and introduced a targeted knockout plasmid (pK18-0948KO) to assess its role in phage adsorption. Our results showed successful transformation and partial segregation, laying the foundation for future functional validation.
Collectively, this work establishes a robust transformation system for Synechococcus WH7803, demonstrates the feasibility of phage DNA delivery via electroporation, and provides new insights into host defense mechanisms.