A Small Molecule Coordinates Symbiotic Behaviors in a Host Organ.

The lifelong relationship between the Hawaiian bobtail squid and its microbial symbiont represents a simplified model system for studying microbiome establishment and maintenance. The bacteria colonize a dedicated symbiotic light organ in the squid, from which bacterial luminescence camouflages the host in a process termed counterillumination. The squid host hatches without its symbionts, which must be acquired from the ocean amidst a diversity of nonbeneficial bacteria, such that precise molecular communication is required for initiation of the specific relationship. Therefore it is likely there are specialized metabolites used in the light organ microenvironment to modulate these processes. To identify small molecules that may influence the establishment of this symbiosis, we used imaging mass spectrometry to analyze metabolite production in with altered biofilm production, which correlates directly to colonization capability in its host. "Biofilm-up" and "biofilm-down" mutants were compared to a wild-type strain, and ions that were more abundantly produced by the biofilm-up mutant were detected. Using a combination of structural elucidation and synthetic chemistry, one such signal was determined to be a diketopiperazine, cyclo(d-histidyl-l-proline). This diketopiperazine modulated luminescence in and, using imaging mass spectrometry, was directly detected in the light organ of the colonized host. This work highlights the continued need for untargeted discovery efforts in host-microbe interactions and showcases the benefits of the squid- system for identification and characterization of small molecules that modulate microbiome behaviors. The complexity of animal microbiomes presents challenges to defining signaling molecules within the microbial consortium and between the microbes and the host. By focusing on the binary symbiosis between and , we have combined genetic analysis with direct imaging to define and study small molecules in the intact symbiosis. We have detected and characterized a diketopiperazine produced by strong biofilm-forming strains that was detectable in the host symbiotic organ, and which influences bacterial luminescence. Biofilm formation and luminescence are critical for initiation and maintenance of the association, respectively, suggesting that the compound may link early and later development stages, providing further evidence that multiple small molecules are important in establishing these beneficial relationships.