A novel, conserved cluster of genes promotes symbiotic colonization and σ⁵⁴‐dependent biofilm formation by Vibrio fischeri

Abstract

SummaryVibrio 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 s s s s 54 interaction domain. This induction depended on s s s s 54, as a mutation in rpoN abolished syp transcription. Primer extension analysis supported the use of putative s s s s 54 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 s s s s 54-dependent response regulator and uses to promote symbiotic colonization.

Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire

“…In addition, several genes responsible for colonization were found in the P1 genome. The syp genes, a group of genes described in V. fischeri that promote symbiotic colonization and biofilm formation via Sigma54 regulation (Yip et al, 2005), and the flp genes for biogenesis of type IV Flp pilus described in Actinobacillus actinomycetemcomitans (Boyd et al, 2008), may represent important loci for the process of coral colonization. In addition, strain-specific genes found in the P1 genomes revealed the importance of phage genes driving intra-species diversification.…”

RscS Functions Upstream of SypG To Control the syp Locus and Biofilm Formation in Vibrio fischeri

“…SypG is predicted to be an RR in the NtrC-like family of 54 -dependent transcriptional activators. Like RscS, multicopy expression of sypG results in enhanced syp transcription and biofilm formation, as measured by glass attachment and pellicle formation (43); however, the pellicles are not as robust as those formed by RscS-overexpressing cells. In addition, the overexpression of SypG does not induce wrinkled-colony formation, another hallmark of RscS overexpression (42).…”

“…This cluster is composed of 18 genes organized into at least five putative operons (A to E, F to H, I to L, M to O, and P to R) (43; E. A. Hussa, K. Geszvain, and K. L. Visick, unpublished data). The syp gene products are predicted to function in polysaccharide synthesis and transport, and most are required for the initiation of symbiotic colonization (43). The overexpression of rscS results in the induction of syp transcription and syp-dependent biofilm formation (42).…”

“…One candidate partner already known to affect syp transcription is SypG, a syp-encoded regulator that is also required for colonization (10,43). SypG is predicted to be an RR in the NtrC-like family of 54 -dependent transcriptional activators.…”

Global discovery of colonization determinants in the squid symbiontVibrio fischeri

“…Forward genetic studies in V. fischeri have proven fruitful for identification of relevant colonization factors. Previous work included identification of Tn10 transposon mutants that individually failed to colonize the squid host; that work identified the RscS (regulator of symbiotic colonization-sensor) biofilm regulator and Syp (symbiosis polysaccharide) biofilm effectors that are required for robust colonization (5,6). Global genetic approaches have been applied in culture to identify regulators of the hallmark luminescence phenotype, of flagellar motility, and in other signaling pathways (7,8).…”