In Salmonella enterica serovar Typhimurium, proteolytic cleavage of the membrane-bound transcriptional regulator CadC acts as a switch to activate genes of the lysine decarboxylase system in response to low pH and lysine signals. To identify the genetic factors required for the proteolytic activation of CadC, we performed genome-wide random mutagenesis. We show that a phosphotransferase system (PTS) permease STM4538 acts as a positive modulator of CadC function. The transposon insertion in STM4538 reduces the expression of the CadC target operon cadBA under permissive conditions. In addition, deletional inactivation of STM4538 in the wild-type background leads to the impaired proteolytic cleavage of CadC. We also show that only the low pH signal is involved in the proteolytic processing of CadC, but the lysine signal plays a role in the repression of the lysP gene encoding a lysine-specific permease, which negatively controls expression of the cadBA operon. Our data suggest that the PTS permease STM4538 affects proteolytic processing, which is a necessary but not sufficient step for CadC activation, rendering CadC able to activate target genes.