UC Merced

All animals have long-term beneficial associations with bacterial microbiomes. The unique mutualism between the bobtail squid Euprymna scolopes and the marine bioluminescent bacterium Vibrio fischeri, is a powerful model system for studies of initiation, establishment, and maintenance of symbiosis. Planktonic V. fischeri encountering a hatchling squid colonize the interior of the squid light organ, where they produce bioluminescence used by the host during its nocturnal foraging.Increasingly sophisticated molecular genetic techniques have been developed to construct mutant strains of V. fischeri that facilitate studies of the mechanisms underlying the symbiosis. Here I report the application of novel techniques to studies of the adaptation of V. fischeri to the changing environmental conditions it encounters within the light organ during symbiosis. First, I constructed a pH-sensitive fluorescent bioreporter strain of V. fischeri which can monitor and report environmental pH. V. fischeri adapts to changes in environmental pH throughout its life-cycle, ranging from neutral seawater to an acidic (pH~5.5) nocturnal light organ interior. I validated the utility of the bioreporter by assaying the acidification of cultured V. fischeri due to aerobic fermentation, analogous to the acidification of the light organ. Next, I developed a CRISPR Interference (CRISPRi) suite of plasmid vectors which enables the inducible, titratable, and reversable repression of targetable genes-of-interest. In CRISPRi, a mutant cas9 protein/sgRNA transcript complex binds to a genomic locus complementary to the sgRNA sequence and blocks the initiation/extension of mRNA transcription via steric hindrance. I demonstrated that this V. fischeri CRISPRi system can inducibly repress expression of an exogenous fluorescent reporter gene (mRFP), as well as the endogenous luxC luminescence and flrA flagellar regulator genes, both singularly and simultaneously. The V. fischeri CRISPRi system was used in studies of colonized squid to show that repression of luminescence led to rejection of the “dim” symbionts 48 hours post infection, a time period not accessible for study using conventional mutant strains. Finally, I investigated natural transformation (NT), the uptake and genomic integration of environmental DNA, in V. fischeri. I observed that experimental NT can be induced under acidic conditions that reflect the pH of the squid nocturnal light organ. I also developed enhanced methodologies for inducing natural transformation with a resident plasmid, rather than a chromosomal, integration site, and show that this method allows for in vivo cloning of V. fischeri. A significant increase in NT frequency was also observed using a novel NT/CRISPRi vector targeting the dns exonuclease gene. These findings provide insight into the adaptation of V. fischeri to the changing environmental conditions in the host light organ, and provide a complement of novel molecular genetic tools which will facilitate further studies of this beneficial symbiosis.