Rahlff, J.; Lang-Yona, N.; Lahav, E.; Westmeijer, G.; Das, R.; Buder, K.; Bueschel, R.; Micheel, J.; Eckhardt, S.; Evangeliou, N.; Groot Zwaaftink, C.; van Pinxteren, M.

Background: Cloud water harbors diverse microbial communities despite its extreme oligotrophic conditions. However, the ecological and evolutionary dynamics of viruses in these transient atmospheric habitats remain poorly understood. Clouds have traditionally been regarded primarily as passive carriers of microorganisms rather than as active ecological environments supporting microbial interactions. In this study, cloud water was sampled at Mount Verde, Cape Verde Islands (744 m a.s.l.). We performed metagenomic analyses of iron-flocculated cloud water alongside genome analyses of a bacterial isolate and metagenome-assembled genomes using established bioinformatic approaches. Viral diversity, virus-host interactions, metabolic functions, genetic adaptations, and viral population dynamics across cloud events were investigated. In addition, UV-B resistance experiments were conducted for a novel cloud-water isolate. Results: We isolated 24 cloud water bacteria, including four novel species lineages, and recovered 62 high-quality metagenome-assembled genomes, including 10 novel species lineages. We identified 458 viral operational taxonomic units and 237 virus-host linkages across diverse prokaryotic hosts, revealing active viral predation across diverse bacterial taxa. In addition, CRISPR spacer matches from isolates of novel bacterial lineages such as Deinococcus nubigenus MPC36 were found. Viruses carried genes involved in host adaptation to environmental stressors, including cold-shock response, UV radiation resistance, and osmotic stress. In addition, viral populations exhibited SNP-level microdiversity and shifts in single-nucleotide variant composition across temporally proximate cloud events, indicating rapid population turnover. Experimental characterization of the cloud isolate Curtobacterium nubigenum MPC39 further revealed pronounced resistance to UV-B radiation and the presence of an inducible prophage, Curtobacterium phage vB_CnuS_Cirrus1 assigned to the new viral family Nebulaviridae, which could be validated in transmission electron microscopy. Reconstructed genomes from cloud-associated bacteria encoded carbon monoxide dehydrogenase genes and UV resistance genes, suggesting trace gas metabolism and enhanced UV protection as survival strategies in oligotrophic cloud droplets. In silico replication rates estimated using iRep were consistent with active bacterial replication at the time of sampling. Conclusions: Together, these findings demonstrate that clouds are not merely passive carriers of microorganisms, but dynamic atmospheric ecosystems in which virus-host interactions shape microbial diversity and contribute to microbial turnover, atmospheric dispersal, and cloud-water biogeochemistry.