Biosynthesis and import of the cytoprotective extremolytes ectoine and hydroxyectoine in the phylum Planctomycetota

Many bacteria inhabiting high-salinity environments accumulate compatible solutes, water-soluble organic compounds that are highly congruent with cellular biochemistry and physiology. This "salt-out" strategy counteracts cytoplasmic water loss, maintains turgor within physiologically acceptable limits, and enables growth under conditions conferring osmotic stress. Ectoine and its derivative hydroxyectoine are prominent compatible solutes that function not only as efficient osmotic stress protectants but also serve as chemical chaperones and cytoprotectants. These properties have driven industrial-scale biotechnological production of ectoines and their broad practical applications. Despite their well-established role in stress protection, the distribution of ectoines within the widespread Planctomycetota has remained largely unexplored. Here, we analyzed the genomes of 163 type strains within this phylum and identified 23 species harboring ectoine biosynthetic gene clusters (ect). These clusters were predominantly present in marine-, saline- and brackish water-associated members of the families Planctomycetaceae and Pirellulaceae, with few representatives present in Lacipirellulaceae. Experimental validation of ect cluster functionality in four Planctomycetota species confirmed increased ectoine and hydroxyectoine production under osmotic stress, supporting their role as extremolytes. All ect clusters co-localize with genes encoding compatible solute transporters from the ABC (EhuABCD), TRAP-T (TeaABC; UheABC), or sodium solute symporter (SSS; EctI) families. Functional characterization of EctI from the type strain Rubinisphaera brasiliensis demonstrated uptake of hydroxyectoine and additional compatible solutes, including proline betaine, homobetaine, glycine betaine, and dimethylsulfoniopropionate (DMSP). Together, our findings reveal a lineage-specific adaptation of Planctomycetota to sustained osmotic stress through both synthesis and uptake of ectoines, providing new insights into the ecophysiology of these microorganisms.