Regulated secretion of YopN by the type III machinery of Yersinia enterocolitica

During infection, Yersinia enterocolitica exports Yop proteins via a type I II secretion pathway. Secretion is activated when the environmental concent ration of calcium ions is below 100 muM (low-calcium response). Yersiniae l acking yopN (lcrE), yscB, sycN, or tyeA do not inactivate the type III path way even when the concentration of calcium is above 100 muM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of yopN gene sequences to th e 5 ' end of the npt reporter generates hybrid proteins that are transporte d by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of yopN. Expression of plasmid-borne yopN, but not of yopN(1-294)-npt, complem ents the calcium-blind phenotype of yopN mutants. Surprisingly, yopN mutant s respond to environmental changes in calcium concentration and secrete Yop N(1-294)-Npt in the absence but not in the presence of calcium. tyeA is req uired for the low-calcium regulation of YopN(1-294)-Npt secretion, whereas sycN and yscB mutants fail to secrete YopN(1-294)-Npt in the presence of ca lcium. Experiments with yopN-npt fusions identified two other signals that regulate the secretion of YopN.yopN codons 16 to 100 prevent the entry of Y opN into the type III pathway, a negative regulatory effect that is overcom e by expression of yscB and sycN. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double m embrane envelope in the presence of functional tyeA. These data support a m odel whereby YopN transport may serve as a regulatory mechanism for the act ivity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environme ntal calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.