Trait-based signatures associated with persistence and thermal benefit in a genomically decayed coral probiotic.

A key bottleneck in microbiome engineering is ensuring long-term host association of introduced microbes. Selecting probiotic candidates based on evolutionary genomic decay signatures of emerging host dependency offers a potential solution. The Ruegeria strain B4 of population MC10, identified by such signatures, showed persistent coral colonization in a companion study. Whether this persistence translates into measurable host benefit compared to other coral-associated Ruegeria strains, and which mechanisms underlie such benefit, remained unknown. Here we directly compare the probiotic efficacy of MC10-B4 against two sympatric Ruegeria strains isolated from the same coral colony and mucus compartment, controlling for host genotype and microenvironment. MC10-B4 inoculation significantly increased heat stress tolerance in the model cnidarian Aiptasia (Exaiptasia diaphana strain H2), outperforming both controls. To understand the mechanistic basis, we characterized the functional profile of MC10-B4 using integrated multi-omics. The MC10 genome is enriched in host-interaction genes, including siderophore-mediated iron acquisition and exopolysaccharide biosynthesis, confirmed phenotypically by iron scavenging and enhanced biofilm formation. Following exposure to coral tissue extract, MC10-B4 underwent a coordinated "motile-to-sessile" proteomic reprogramming, downregulating flagellar motor components while upregulating flagellin and biofilm regulators. This response was distinct from that of sympatric relatives, which instead mounted a broad upregulation of nutrient acquisition systems. MC10-B4's functional profile, particularly its oxidative stress sensitivity, contrasts with traits favored in conventional probiotic screens. Our results provide mechanistic insight into traits associated with long-term host association and thermal benefit, validating an evolution-guided approach that prioritizes innate colonization potential over pre-defined laboratory functionalities for rational probiotic design.