Previously, it was shown that optimal functioning of the Yersinia type III secretion system (T3SS) in cell culture infection assays requires the exoribonuclease polynucleotide phosphorylase (PNPase) and that normal T3SS activity could be restored in the Deltapnp strains by expressing just the approximately 70-aa S1 RNA-binding domain of PNPase. Here, it is shown that the Yersinia Deltapnp strain is less virulent in the mouse compared with the isogenic wild-type strain. To begin to understand what could be limiting T3SS activity in the absence of PNPase, T3SS-encoding transcripts and proteins in the YersiniaDeltapnp strains were analyzed. Surprisingly, it was found that the Deltapnp Yersinia strains possessed enhanced levels of T3SS-encoding transcripts and proteins compared with the wild-type strains. We then found that an S1 variant containing a disruption in its RNA-binding subdomain was inactive in terms of restoring normal T3SS activity. However, T3SS expression levels did not differ between Deltapnp strains expressing active and inactive S1 proteins, further showing that T3SS activity and expression levels, at least as related to PNPase and its S1 domain, are not linked. The results suggest that PNPase affects the expression and activity of the T3SS by distinct mechanisms and that the S1-dependent effect on T3SS activity involves an RNA intermediate.