Meeting Abstract
The bioluminescent marine bacterium Vibrio fischeri has been used to study mechanisms of environmental specificity in mutualistic associations with animal hosts. V. fischeri colonizes the light organ of sepiolid squids (Cephalopoda: Sepiolidae) and produces luciferase-based light which provides ventral counter-shading camouflage for the squid. Low pH levels may cyclically develop within the colonized light organ as a result of diurnal V. fischeri growth. Adaptation to such stressful conditions may provide V. fischeri a competitive advantage in colonizing the light organ. To investigate this phenomenon, we are developing novel methods of directly assaying pH within the colonized light organ of the Hawaiian bobtail squid, Euprymna scolopes. Previously developed V. fischeri strains containing a pH responsive ratiometric GFP derivative (pHLuorin) have shown promise as a fluorescent pH biosensor enabling the determination of both cytoplasmic and extracellular pH in vitro, but have constrained utility for assaying pH within the light organ due to the necessity of using uncommon fluorescence microscopy excitation/emission filter sets. To negate this limitation, new strains have been developed using a ratiometric mCherry-pHLuorin fusion protein in lieu of the standard pHluorin protein. These can be used to assay pH via fluorescence spectrometry, microscopy, and flow cytometry using standard red/green filter sets, while providing equivalent levels of sensitivity. The use of these novel biosensors in the lumen of the light organ will allow us to determine the extent of cyclic pH fluctuations as a determining factor in establishing successful colonization by V. fischeri in sepiolid squids.
