The Synthesis and Assembly of a Truncated Cyanophage Genome and Its Expression in a Heterogenous Host

Cyanophages play an important role in regulating the dynamics of cyanobacteria communities in the hydrosphere, representing a promising biological control strategy for cyanobacterial blooms. Nevertheless, most cyanophages are host-specific, making it difficult to control blooming cyanobacteria via single or multiple cyanophages. In order to address the issue, we explore the interaction between cyanophages and their heterologous hosts, with the aim of revealing the principles of designing and constructing an artificial cyanophage genome towards multiple cyanobacterial hosts. In the present study, we use synthetic biological approaches to assess the impact of introducing a fragment of cyanophage genome into a heterologous cyanobacterium under a variety of environmental conditions. Based on a natural cyanophage A-4L genome (41,750 bp), a truncated cyanophage genome Syn-A-4-8 is synthesized and assembled in Saccharomyces cerevisiae. We found that a 351-15,930 bp area of the A-4L genome has a fragment that is lethal to Escherichia coli during the process of attempting to assemble the full-length A-4L genome. Syn-A-4-8 was successfully introduced into E. coli and then transferred into the model cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942) via conjugation. Although no significant phenotypes of Syn7942 carrying Syn-A-4-8 (LS-02) could be observed under normal conditions, its growth exhibited a prolonged lag phase compared to that of the control strain under 290-millimolar NaCl stress. Finally, the mechanisms of altered salt tolerance in LS-02 were revealed through comparative transcriptomics, and ORF25 and ORF26 on Syn-A-4-8 turned out to be the key genes causing the phenotype. Our research represents an important attempt in designing artificial cyanophages towards multiple hosts, and offers new future insights into the control of cyanobacterial blooms.