The marine bioluminescent bacterium V. fischeri inhabits a specialized organ in the Hawaiian Bobtail squid Euprymna scolopes called the light organ. A short time after hatching, juvenile E. scolopes squid are rapidly and specifically colonized by the bacterium, establishing a symbiotic relationship that persists throughout the lifetime of the squid. As seawater is vented through the mantle cavity of the squid, cells of V. fischeri present in the water become embedded in mucus secreted from the light organ. The cells then enter into the pores of the light organ where they ultimately navigate to and colonize deep crypts and produce light via bioluminescence. Although it is known that bacterial cells unable to swim cannot colonize juvenile squid, the question of whether or not the ability of the bacterium to sense and move toward an attractant (chemotaxis) is required is unknown. Previously the PI has shown that V. fischeri migrates toward N-acetylneuraminic acid (NANA), a glycan sugar component of the squid light organ mucus. The inability to migrate toward NANA by other marine bacterial Vibrio species suggests that the attraction toward NANA may be specific to V. fischeri. It has also been shown that V. fischeri migrates toward various sugars and components of DNA (nucleosides). Bacteria sense attractants in their environment through the binding of the attractant to receptors in the bacterium's membrane (known as methyl-accepting chemotaxis proteins or MCPs). Upon binding an attractant, the MCPs relay signals to the bacterium's flagella, directing the bacterium to move toward higher concentrations of attractant. E. coli has five such proteins that have been well characterized for structure as well as recognized attractants. Based on the recently-sequenced genome of V. fischeri, there appears to be at least 34 proteins in the bacterium which have strong similarities to sequences common to all MCP proteins (and are therefore putative MCPs). However, only two of these proteins show significant resemblance to specific E. coli MCPs, suggesting that the remaining proteins may recognize attractants distinct from those recognized by E. coli. This study will begin to examine which of the putative V. fischeri MCPs are receptors for known attractants. To accomplish this, molecular biology techniques will be employed to clone the putative MCPs and construct non-functional versions of the proteins (resulting in V. fischeri MCP mutants). These mutants will then be tested for the ability to swim toward various known attractants to identify which MCPs bind specific attractants. In a separate study, the MCP mutants will be tested for the ability to colonize juvenile squid. These studies will aid in understanding how the symbiotic relationship between bacterium and squid is established. Various aspects of this project will also be incorporated into teaching labs in a microbiology course taught by the PI at the University of Southern Indiana.
