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Summary

Vibrio fischeri is the exclusive symbiont residing in the light organ of the squid Euprymna scolopes. To understand the genetic requirements for this association, we searched a library of V. fischeri transposon insertion mutants for those that failed to colonize E. scolopes. We identified four mutants that exhibited severe defects in initiating colonization. Sequence analysis revealed that the strains contained insertions in four different members of a cluster of 21 genes oriented in the same direction. The predicted gene products are similar to proteins involved in capsule, exopolysaccharide or lipopolysaccharide biosynthesis, including six putative glycosyltransferases. We constructed mutations in five additional genes and found that they also were required for symbiosis. Therefore, we have termed this region syp, for symbiosis polysaccharide. Homologous clusters also exist in Vibrio parahaemolyticus and Vibrio vulnificus, and thus these genes may represent a common mechanism for promoting bacteria–host interactions. Using lacZ reporter fusions, we observed that transcription of the syp genes did not occur under standard laboratory conditions, but could be induced by multicopy expression of sypG, which encodes a response regulator with a predicted σ54 interaction domain. This induction depended on σ54, as a mutation in rpoN abolished syp transcription. Primer extension analysis supported the use of putative σ54 binding sites upstream of sypA, sypI and sypM as promoters. Finally, we found that multicopy expression of sypG resulted in robust biofilm formation. This work thus reveals a novel group of genes that V. fischeri controls through a σ54-dependent response regulator and uses to promote symbiotic colonization.