Discovery of the symbiosis polysaccharide (syp) gene cluster of Vibrio fischeri and its role in biofilm formation and symbiosis.

摘要

The bioluminescent bacterium Vibrio fischeri forms a cooperative association with the Hawaiian squid Euprymna scolopes . The colonization of light organ is specific to V. fischeri , therefore, this one host-one symbiont relationship serves as a model for studying host-microbe interactions. I screened a transposon mutant library for bacterial factors that are required for symbiosis and found a mutant that failed to colonize the squid. The mutation mapped to a gene within a cluster of eighteen ORFs which exhibited similarity to those involves in polysaccharide biosynthesis. Therefore, we termed this region the symbiosis polysaccharide (syp) cluster.;I hypothesized that the syp cluster modifies a bacterial surface molecule(s) that is required during symbiosis. We showed that the syp cluster could be induced in culture through multicopy expression of two-component regulators such as sypG and rscS . Wild-type V. fischeri overexpressing syp exhibited biofilm-associated phenotypes. To further understand the role of syp in biofilm formation, I analyzed bacteria by confocal microscopy and found that biofilms of the syp-overexpressing cells were substantially thicker, thus supporting my hypothesis that Syp modifies the cell surface.;Electron microscopy analysis revealed that overexpression of syp resulted in a polysaccharide matrix present both on the surface and between cells within bacterial colonies. Analysis with lectins suggested that this extracellular matrix is composed of, in part, glucose and/or alpha-linked mannose. In addition, these polysaccharides exhibited hydrophobic properties, potentially due to alterations in the composition or structure of the surface polysaccharides. Furthermore, confocal microscopy showed that rscS1 -overexpression enhanced bacterial aggregation in the light organ. To assess the functions of the syp genes, syp mutants were evaluated for their role in biofilm formation and polysaccharide production. Consistent with the predicted functions of the syp cluster, the formation of biofilm associated phenotypes were largely dependent on the syp genes. Thus, this work advances our understanding of the interactions between V. fischeri and squid by defining a locus involved in cell-cell adhesion that promotes the aggregation stage of colonization. The clear correlation between biofilm phenotypes in culture and biofilm-like symbiotic aggregation also provides an advance for the field of biofilm formation.