Optimization of a minimal sample preparation protocol for imaging mass spectrometry of unsectioned juvenile invertebrates.

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.

Nitric oxide inhibits biofilm formation by Vibrio fischeri via the nitric oxide sensor HnoX.

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, the 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 biofilm and a mechanism for the 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.