Expression and characterization of the complete cyanophage genome PP in the heterologous host Synechococcus elongatus PCC 7942

Cyanophages are considered a promising biological management option for treating cyanobacterial blooms. Broadening the host range of cyanophages and/or shortening the lysis cycle by designing and synthesizing artificial cyanophages are potential strategies to enhance their effectiveness and efficiency. However, the rescue of artificial cyanophage genomes remains unexplored. In this study, we achieved the integration of a full-length cyanophage genome, PP, which originally infects Plectonema boryanum FACHB-240, into the model cyanobacterium Synechococcus elongatus PCC 7942. Since the integration of these large fragments (∼42 kb) into cyanobacteria depended on conjugation via Escherichia coli , the toxic open reading frames (ORFs) of PP to E. coli were first identified, leading to the identification of toxic ORF6, ORF11, and ORF22. The original PP genome was then rearranged, and the three toxic ORFs were controlled using a tandem induction switch. The full length of the PP genome was integrated into the genome of S. elongatus PCC 7942 via two rounds of homologous recombination.

Interestingly, compared to the control strain, the integration of the PP genome decreased photosynthesis and carbon fixation in S. elongatus PCC 7942, exhibiting cyanophage-like behavior. Transcriptomic analysis revealed that 32 of the 41 ORFs of the PP genome were transcribed in S. elongatus PCC 7942, significantly altering the energy metabolism and carbon fixation pathways. These influences were further demonstrated using metabolomics. This study provides a comprehensive approach for the artificial design and integration of cyanophage genomes in cyanobacteria, laying the foundation for their real rescue in the future.