The sea-surface microlayer (SML) represents a unique and challenging environment between the atmosphere and the ocean, covering ~70% of the Earth’s surface with a thickness of 1 to 1000 μm. This layer is distinct in its physico-chemical and biological properties compared to underlying waters and is influenced by extreme environmental factors. Therefore, microbial life must adapt to harsh conditions. In this study, we isolated and characterized bacteriophages from four Flavobacteria strains collected from Baltic Sea SML, sea foam, and beach foam offshore Öland, Sweden.
We performed host-range analysis and whole-genome sequencing of 45 phages and studied representative ones in transmission electron microscopy (TEM). Genome clustering in VipTree revealed protein similarities of the isolates to known Flavobacteria and Cellulophaga phages from the Baltic Sea. We identified eleven phage genera and 13 species clusters. According to transmission electron microscopy analysis, three, two and five phage species clusters corresponded to podo-, myo-, and siphophage morphologies in TEM, respectively. Host-range analysis of 61 phages isolated from 4 hosts revealed that only one podophage isolate could cross-infect two hosts, while all other phages were host-specific. Notably, twelve different phages belonging to four species cluster, originally isolated on two of the hosts, carried a gene functionally annotated as insect antifreeze protein (AFP), suggesting a possible role in enhancing bacterial survival in cold conditions or freezing of the water surface. Alphafold prediction revealed superposition of the phage AFP to the AFP structure of the yellow mealworm beetle Tenebrio molitor, showing highly conserved cysteines. A BLASTp of the AFP protein sequence against the Ocean Gene Atlas showed its wide spatial distribution in TARA Ocean metagenomes up to 31°C. However, in metatranscriptomes the distribution was limited to subpolar regions with highest abundance around 7°C, suggesting expression at low temperatures. Our results show that air-sea interface ecosystems are a reservoir of Flavobacteria bacteriophages with narrow host ranges, displaying important environmental adaptations.

Bacteriophage,Sea surface microlayer,Foams,Flavobacteria,Antifreeze Protein,Adaptation,Baltic Sea