Transient infection of <i>Euprymna scolopes</i> with an engineered D-alanine auxotroph of <i>Vibrio fischeri</i>

The symbiosis between <i>Vibrio fischeri</i> and the Hawaiian bobtail squid, <i>Euprymna scolopes</i>, is a tractable and well-studied model of bacteria-animal mutualism. Here, we developed a method to transiently colonize <i>E. scolopes</i> 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 (<i>alr</i>) mutants of <i>V. fischeri</i> that lack avenues for mutational suppression of auxotrophy or reversion to prototrophy. Surprisingly, an ∆<i>alr</i> mutant did not require D-ala to grow in a minimal medium, a phenomenon requiring <i>metC</i>, which encodes cystathionine β-lyase. Likewise, overexpression of <i>metC</i> suppressed D-ala auxotrophy in a rich medium. To block potential mechanisms of suppression, we combined the ∆<i>alr</i> mutation with deletions of <i>metC</i> and/or <i>bsrF</i>, which encodes a broad-spectrum racemase and investigated the suppression rates of four D-ala auxotrophic strains. We then focused on ∆<i>alr</i> ∆<i>bsrF</i> 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 <i>E. scolopes</i> 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 <i>E. scolopes</i> light organ.IMPORTANCEThe importance of this study is multi-faceted, providing a valuable methodological tool and insight into the biology of the symbiosis between <i>Vibrio fischeri</i> and <i>Euprymna scolopes</i>. First, the study sheds light on the critical role of D-ala for bacterial growth, and the underpinnings of D-ala synthesis. Our observations that <i>metC</i> obviates the need for D-ala supplementation of an <i>alr</i> mutant in minimal medium and that MetC-dependent growth correlates with D-ala in peptidoglycan, corroborate and extend previous findings in <i>Escherichia coli</i> regarding a role of MetC in D-ala production. Second, our isolation of robust D-ala auxotrophs led us to a novel method for studying the squid-<i>Vibrio</i> symbiosis, allowing for transient colonization without the use of antibiotics, and revealed intriguing differences in symbiont growth parameters in distinct light organ crypts. This work and the methodology developed will contribute to our understanding of the persistence and dynamics of <i>V. fischeri</i> within its host.