Abstract:

Marine cyanobacteria Synechococcus are pivotal contributors to global carbon fixation, yet their defense mechanisms against cyanophage predation remain poorly characterized. Unlike heterotrophic bacteria, almost all marine Synechococcus lack CRISPR–Cas systems, and most also lack restriction–modification systems. This absence highlights the likelihood of unique defense mechanisms in marine Synechococcus. Earlier work isolated spontaneous phage-resistant Synechococcus WH7803 mutant strains (WH7803-4a1, -4a2, -4a3, and -4a4) through selective pressure using cyanophage S-SCSM4a. Subsequent comparative genomic analysis identified three mutant genes (SynWH7803_2273, SynWH7803_0948, and SynWH7803_2298) in these mutant strains. In this study, we investigate phage resistance mechanisms in Synechococcus WH7803, focusing on these four mutant strains carrying their respective mutations. Phage adsorption assays revealed that the cyanophage failed to attach to the mutant strains, suggesting that the identified mutations may alter surface receptor conformation or membrane properties critical for phage binding. Genomic screening further identified a putative type II TA system (SynWH7803_1944/1945) in Synechococcus WH7803 genome, which was hypothesized to mediate antiphage defense. To investigate the roles of the three mutant genes and the predicted TA system in Synechococcus WH7803 in conferring phage resistance, we employed genome editing techniques to systematically knockout each target. We constructed plasmids based on the suicide vector pK18mobsacB and transformed them individually for targeted gene inactivation via homologous recombination. Subsequently, we will screen for successful knockout mutants and perform phage susceptibility assays to validate their roles in conferring phage resistance. This study will uncover novel defense mechanisms in marine cyanobacteria, bridging a critical gap in understanding phage-host dynamics.

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