The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization

Curli are functional amyloid fibers assembled by enteric bacteria such asEscherichia coli andSalmonella spp. InE. coli , the polymerization of the major curli fiber subunit protein CsgA into an amyloid fiber depends on the minor curli subunit protein, CsgB. The outer membrane-localized CsgB protein shares ≈30% sequence identity with the amyloid-forming protein CsgA, suggesting that CsgB might also have amyloidogenic properties. Here, we characterized the biochemical properties of CsgB and the molecular basis for CsgB-mediated nucleation of CsgA. Deletion analysis revealed that a CsgB molecule missing 19 amino acids from its C terminus (CsgBtrunc ) was not outer membrane-associated, but secreted away from the cell. CsgBtrunc was overexpressed and purified from the extracellular milieu of cells as an SDS-soluble, nonaggregated protein. Soluble CsgBtrunc assembled into fibers that bound to the amyloid-specific dyes Congo red and thioflavin-T. CsgBtrunc fibers were able to seed soluble CsgA polymerizationin vitro . CsgBtrunc displayed modest nucleator activityin vivo , as demonstrated by its ability to convert extracellular CsgA into an amyloid fiber. Unlike WT CsgB, CsgBtrunc was only able to act as a nucleator when cells were genetically manipulated to secrete higher concentrations of CsgA. This work represents a unique demonstration of functional amyloid nucleation and it suggests an elegant model for howE. coli guides efficient amyloid fiber formation on the cell surface.