Transient infection of Euprymna scolopes with an engineered D-alanine auxotroph of Vibrio fischeri

Abstract

The symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes, is a tractable and well-studied model of bacteria-animal mutualism. Here, we developed a method to transiently colonize E. scolopes using D-alanine (D-ala) auxotrophy of the symbiont, controlling the persistence of viable infection by supplying or withholding D-ala. We generated alanine racemase (alr) mutants of V. fischeri that lack avenues for mutational suppression of auxotrophy or reversion to prototrophy. Surprisingly, an ∆alr mutant did not require D-ala to grow in a minimal medium, a phenomenon requiring metC, which encodes cystathionine β-lyase. Likewise, overexpression of metC suppressed D-ala auxotrophy in a rich medium. To block potential mechanisms of suppression, we combined the ∆alr mutation with deletions of metC and/or bsrF, which encodes a broad-spectrum racemase and investigated the suppression rates of four D-ala auxotrophic strains. We then focused on ∆alr ∆bsrF mutant MC13, which has a suppression rate of <10⁻⁹. When D-ala was removed from a growing culture of MC13, cells rounded and lysed within 40 minutes. Transient colonization of E. scolopes was achieved by inoculating squid in seawater containing MC13 and D-ala, and then transferring the squid into water lacking D-ala, which resulted in loss of viable symbionts within hours. Interestingly, the symbionts within crypt 3 persisted longer than those of crypt 1, suggesting a difference in bacterial growth rate in distinct crypt environments. Our study highlights a new approach for inducing transient colonization and provides insight into the biogeography of the E. scolopes light organ.

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