Two distinct bacterial biofilm components trigger metamorphosis in the colonial hydrozoanHydractinia echinata

In the marine environment bacterial-induced metamorphosis of larvae is a widespread cross-kingdom communication phenomenon and critical for the persistence of many marine invertebrates. However, the identities of most inducing bacterial signals and the underlying cellular mechanisms remain enigmatic. Larvae of Hydractinia echinata provide an excellent model for investigating bacteria-stimulated settlement as they transform upon detection of the signal into the colonial adult stage within 24 h. Although H. echinata served as cell biological model system for decades, the influence of bacterial signals on the morphogenic transition remained largely unexplored. Using a bioassay-guided analysis, we first identified that specific bacterial (lyso)phospholipids, naturally present in bacterial biofilms, elicit metamorphosis in Hydractinia larvae in a dose-response matter. In particular, lysophospholipids as single compounds or in combinations at 50 µM concentrations induced metamorphosis in up to 50% of all larvae phospholipid within 48 h. By using fluorescence-labeled bacterial phospholipids, we demonstrated their incorporation into the larval membranes, where interactions with internal signaling cascades could occur. In addition, two structurally distinct exopolysaccharides, the newly identified Rha-Man polysaccharide from Pseudoalteromonas sp. P1-9 and curdlan from Alcaligenes faecalis caused up to 75% of all larvae to transform within 24 h. We also found that combinations of (lyso)phospholipids and curdlan induced the transformation in almost all larvae within 24 h, thereby exceeding the morphogenic activity observed for single compounds and axenic bacterial biofilms. Our results demonstrate that multiple and structurally distinct bacterial-derived metabolites converge to induce high transformation rates of Hydractinia larvae, which might ensure optimal habitat selection despite the general widespread occurrence of both compound classes. Significance Statement Bacterial biofilms profoundly influence the recruitment and settlement of marine invertebrates, critical steps for diverse marine processes such as coral reef formation, marine fisheries and the fouling of submerged surfaces. Yet, the complex composition of biofilms often makes it challenging to characterize the individual signals and regulatory mechanisms. Developing tractable model systems to characterize these co-evolved interactions is the key to understand fundamental processes in evolutionary biology. Here, we characterized for the first time two types of bacterial signaling molecules that induce the morphogenic transition and analyzed their abundance and combinatorial activity. This study highlights the crucial role of the converging activity of multiple bacterial signals in development-related cross-kingdom signaling. Areas Major: Chemical Biology, Microbiology, Developmental Biology