The Ser/Thr protein kinase YpkA, encoded by the virulence plasmid pIB1, is an indispensable virulence determinant of Yersinia pseudotuberculosis [E. E. Galyov, S. Håkansson, A. Forsberg, and H. Wolf-Watz, Nature (London) 361:730-732, 1993]. In this study, the organization of the ypkA-containing operon and the in vitro regulation of this transcriptional unit were characterized. The operon contains two structural genes, ypkA and yopJ, and is regulated by temperature and the extracellular concentration of Ca2+, as are the yop genes. The two proteins were secreted without posttranslational processing, showing that YpkA and YopJ belong to the Yop family. Mutational analysis revealed that, in contrast to all other Yop proteins so far studied, the YopJ protein was dispensable for virulence of Y. pseudotuberculosis.