Nitric oxide (NO) is an important defense molecule secreted by the squid Euprymna scolopes and sensed by the bacterial symbiont, Vibrio fischeri, via the NO-sensor HnoX. HnoX inhibits colonization through an unknown mechanism. The genomic location of hnoX adjacent to hahK, a recently identified positive regulator of biofilm formation, suggested that HnoX may inhibit colonization by controlling biofilm formation, a key early step in colonization. Indeed, deletion of hnoX resulted in early biofilm formation in vitro, an effect that was dependent on HahK and its putative phosphotransfer residues. An allele of hnoX that encodes a protein with increased activity severely delayed wrinkled colony formation. Control occurred at the level of transcription of the syp genes, which produce the polysaccharide matrix component. The addition of NO abrogated biofilm formation and diminished syp transcription, effects that required HnoX. Finally, an hnoX mutant formed larger symbiotic biofilms. This work has thus uncovered a host-relevant signal controlling biofilms and a mechanism for inhibition of biofilm formation by V. fischeri. The study of V. fischeri HnoX permits us to understand not only host-associated biofilm mechanisms, but also the function of HnoX domain proteins as regulators of important bacterial processes.

Tissue sections have long been the subject matter for the application of imaging mass spectrometry, but recently the technique has been adapted for many other purposes including bacterial colonies and 3D cell culture. Here, we present a simple preparation method for unsectioned invertebrate tissue without the need for fixing, embedding, or slicing. The protocol was used to successfully prepare a Hawaiian bobtail squid hatchling for analysis, and the resulting data detected ions that correspond to compounds present in the host only during its symbiotic colonization by Vibrio fischeri.

The lifelong relationship between the Hawaiian bobtail squid, Euprymna scolopes, and its microbial symbiont, Vibrio fischeri, 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 hosts in a process termed counterillumination. The squid hosts hatch without their symbionts, which must be acquired from the ocean amid a diversity of non-beneficial bacteria, so precise molecular communication is required for initiation of the specific relationship. It is therefore likely that there may be 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 V. fischeri 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 masses that were more abundantly produced by the biofilm-up mutant were detected. Using a combination of structure elucidation and synthetic chemistry, one such signal was determined to be a diketopiperazine, cyclo(D-histidyl-L-proline). This diketopiperazine modulated luminescence in V. fischeri and, using label-free 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-Vibrio system for identification and characterization of small molecules that modulate microbiome behaviors.