Nitrogen availability shapes evolution of phage resistance in cyanobacteria.

Nitrogen-fixing cyanobacteria play a key role in nitrogen and carbon biogeochemical cycles in aquatic ecosystems. Under nitrogen-limited conditions, their ability to fix nitrogen provides an advantage over other species and enables them to form harmful blooms, which are increasing in frequency and negatively impact aquatic environments. Cyanophages (viruses infecting cyanobacteria) impose strong selective pressures on these populations, and although cyanobacteria can rapidly evolve resistance to these phages, there is a tradeoff between phage resistance and nitrogen fixation. Therefore, it remains unclear whether nitrogen-fixing cyanobacteria can evolve resistance without compromising nitrogen fixation under bloom-inducing nitrogen starvation. Here, we explore the evolution of phage resistance in nitrogen-fixing cyanobacteria (Nostoc sp. strain PCC 7120 and Cylindrospermopsis raciborskii) under nitrogen starvation. We found that phage-resistant strains evolved under nitrogen starvation, although resistance emerged more slowly than in nitrogen-rich environments. Whole-genome sequencing of 34 resistant strains revealed that mutations conferring resistance differed between nitrogen-rich and nitrogen-starved conditions. Nitrogen starvation selected for mutations predominantly in glycosyltransferase genes, which are associated with cell surface modifications. In contrast to resistant strains isolated under nitrogen-replete conditions, which exhibited impaired heterocyst formation, resistant strains selected under nitrogen starvation maintained their ability to form functional heterocysts and persist in nitrogen-limited environments. Our findings suggest that nitrogen availability influences the evolutionary trajectory of phage resistance, favoring mechanisms compatible with nitrogen fixation under nitrogen starvation. These results provide new insights into the ecological resilience of nitrogen-fixing cyanobacteria under phage predation and demonstrate that nitrogen availability affects the cost of resistance, evolutionary trajectories, and resistance mechanisms.