ABSTRACTDNA methylation by the DNA adenine methyltransferase (Dam) interferes with the coordinated expression of virulence functions in an increasing number of pathogens. While analyzing the effect of Dam on the virulence of the human pathogenYersinia enterocolitica, we observed type III secretion of Yop effector proteins under nonpermissive conditions. Dam alters the Ca2+regulation of Yop secretion but does not affect the temperature regulation of Yop/Ysc expression. The phenotype is different from that of classical “Ca2+-blind” mutants ofYersinia, as Dam-overproducing (DamOP) strains still translocate Yops polarly into eukaryotic cells. Although transcription of thelcrGVandyopN-tyeAoperons is slightly upregulated, LcrG is absent from lysates of DamOPbacteria, while the amounts of YopN and TyeA are not changed. We present evidence thatclpXPexpression increases after Dam overproduction and that the ClpP protease then degrades LcrG, thereby releasing a block in type III secretion. This is the first example of posttranslational regulation of type III secretion by the Clp protease and adds a new flavor to the complex regulatory mechanisms underlying the controlled release of effector proteins from bacterial cells.