FIG. 5.

DC3000 hrpJ mutant maintains the ability to secrete HrpA1 and AvrPto1 in culture but cannot secrete detectable amounts of HrpZ1. DC3000 strains were grown in type III-inducing conditions, and secretion assays were performed to determine whether natively expressed TTSS substrates were secreted in culture. The strains used were as follows: wild-type DC3000, a DC3000 hrcC mutant defective in TTSS, the DC3000 hrpJ mutant UNL140, and UNL140 carrying pLN726, which contains hrpJ. The cultures were separated into cell-bound and supernatant fractions, and these were subjected to SDS-PAGE and immunoblot analysis. HrpA1, HrpZ1, and AvrPto1 were detected with anti-HrpA1, -HrpZ1, and -AvrPto1 antibodies, respectively. Each strain also contained pCPP2318, which encodes β-lactamase (β-Lac) lacking its export sequence and therefore remains cell bound unless significant nonspecific cell lysis occurs. Reduced levels of HrpA1 and AvrPto1 were detected in hrpJ supernatant fractions. In contrast, HrpZ1 was not detected in supernatant fractions of the hrpJ mutant.