Comparative genomics and super-resolution microscopy of novel cyanophages reveals genomic isomerism, auxiliary genes, and a non-synchronous infection process

Background Microbial communities of multicellular, heterocyst-forming cyanobacteria assimilate organic nitrogen from dinitrogen gas (N ₂ ) through nitrogen fixation and are important photosynthetic primary producers. This makes them ecologically significant, particularly in natural freshwater bodies, wetlands, and agricultural ecosystems. Cyanophages, viruses that specifically infect cyanobacteria play a key role in lysing them, terminating cyanobacterial blooms, and turning over the nutrients accumulated by these communities, with tremendous relevance for the biogeochemical cycles in these environments. However, only a limited number of cyanophages infecting N ₂ -fixing cyanobacteria have been genome sequenced to date, and it has remained unknown how their interaction proceeds with the individual cells within the filaments forming the backbone of these communities. Results We isolated and characterized three Caudoviricetes cyanophages (A-Lf14, A-Alj1 and A-Hlh1) specifically infecting the N ₂ -fixing cyanobacterium Anabaena sp. PCC 7120. Super-resolution microscopy revealed heterogeneous infection outcomes among adjacent cells within individual host filaments, demonstrating cell-level specificity in host defense. Comparative genomics revealed a conserved ~ 30 kb invertible genomic region flanked by inverted repeats, a structural configuration not previously recognized in cyanophages. Using diagnostic PCR, Sanger sequencing, and read-mapping, we validated that two isomers stably coexist within phage populations. In contrast to the previously characterized cyanophage A-1(L), the three new phages encode a non-cyanobacterial alkaline phosphatase (PhoD). The expression of phoD was highly upregulated during infection, suggesting a phage-driven mechanism for phosphorus acquisition. Conversely, these phages lack the early-expressed tnpB endonuclease gene carried by A-1(L). Transcription of tnpB was strongly upregulated for both strands ( tnpB _fwd and tnpB _rev ) during A-1(L) infection, while infection by the three novel phages modestly induced the transcription of five identical host-encoded tnpB copies. In a highly unusual setting, both tnpB strands are protein-coding over their entire length, leading to the expression of a 347-residue protein from the tnpB _rev strand. Conclusions This study reveals that genomic isomerism is a source of structural plasticity in freshwater cyanophages and uncovers functionally distinct auxiliary gene repertoires that link phage infection strategies to nutrient status. Our findings provide mechanistic insight into how cyanophages diversify their genomes, interact with their hosts and show that a phage gene can be protein-coding on both strands.