SsrA/SsrB is a primary two-component system that mediates the survival and replication of Salmonella within host cells. When activated, the SsrB response regulator directly promotes the transcription of multiple genes within Salmonella pathogenicity island 2 (SPI-2). As expression of the SsrB protein is promoted by several transcription factors, including SsrB itself, the expression of SPI-2 genes can increase to undesirable levels under activating conditions. Here, we report that Salmonella can avoid the hyperactivation of SPI-2 genes by using ptsN-encoded EIIA Ntr , a component of the nitrogen-metabolic phosphotransferase system. Under SPI-2-inducing conditions, the levels of SsrB-regulated gene transcription increased abnormally in a ptsN deletion mutant, whereas they decreased in a strain overexpressing EIIA Ntr . We found that EIIA Ntr controls SPI-2 genes by acting on the SsrB protein at the posttranscriptional level. EIIA Ntr interacted directly with SsrB, which prevented the SsrB protein from binding to its target promoter. Finally, the Salmonella strain, either lacking the ptsN gene or overexpressing EIIA Ntr , was unable to replicate within macrophages, and the ptsN deletion mutant was attenuated for virulence in mice. These results indicated that normal SPI-2 gene expression maintained by an EIIA Ntr -SsrB interaction is another determinant of Salmonella virulence.virulence gene regulation | nitrogen-metabolic phosphotransferase system (PTS) | protein-protein interaction