RESUMEN

Cyanophages are viruses that specifically infect cyanobacteria and are capable of regulating the population densities and seasonal distributions of cyanobacteria. However, few studies have investigated the interactions between cyanophages and heterologous hosts, owing to the inability of cyanophages to infect heterologous cyanobacterial hosts. Here, a truncated artificial cyanophage genome, Syn-P4-8, was designed and assembled that contained 18 genes for viral coat assembly proteins but not genes related to host infection or DNA replication. Syn-P4-8 was transferred into the heterologous host Synechocystis sp. PCC 6803 by conjugation. The growth of strain CS-02 carrying Syn-P4-8 was significantly better than that of the control strain when grown in medium containing 5% NaCl. Only two cyanophage genes, encoding the tail protein (open reading frame 25 [ORF25]) and the tail fiber protein (ORF26), were transcribed in Synechocystis PCC 6803 grown in BG11 medium supplemented with 5% NaCl. However, expression of either ORF25 or ORF26 alone could not recover this phenotype. In addition, transcriptomic analysis revealed the presence of 334 differentially expressed genes in CS-02 compared to the control strain, corresponding to 151 downregulated and 183 upregulated genes that may affect cyanobacterial salt tolerances. In this study, synthetic biology methods were used to strengthen our understanding of the interactions between cyanophage genes and heterologous hosts. IMPORTANCE We synthesized and assembled a truncated cyanophage genome called Syn-P4-8, containing 18 genes for viral coat assembly proteins, and transferred it into a nonhost strain, Synechocystis sp. PCC 6803, to investigate interactions between Syn-P4-8 and Synechocystis PCC 6803. We found that coexpression of tail fiber and tail protein genes enhanced the salt tolerance of Synechocystis PCC 6803.

Asunto(s)

Synechocystis , Synechocystis/genética , Synechocystis/metabolismo , Tolerancia a la Sal/genética , Cloruro de Sodio/metabolismo , Proteínas de la Cápside/metabolismo , Genómica

RESUMEN

Artificial cyanophages are considered to be an effective biological method to control harmful cyanobacterial bloom. However, no synthetic cyanophage genome has been constructed and where its obstacles are unclear. Here, we survey a stretch of 16 kb length sequence of cyanophage A-4L that is unclonable in Escherichia coli. We test 12 predicted promoters of cyanophage A-4L which were verified all active in E. coli. Next, we screen for eight ORFs that hindered the assembly of intermediate DNA fragments in E. coli and describe that seven ORFs in the 16 kb sequence could not be separately cloned in E. coli. All of unclonable ORFs in high-copy-number plasmid were successfully cloned using low-copy-number vector, suggesting that these ORFs were copy-number-dependent. We propose a clone strategy abandoned the promotor and the start codon that could be applied for unclonable ORFs. Last, we de novo synthesized and assembled the full-length genome of cyanophage A-4L. This work deepens the understanding of synthetic cyanophages studies.