1a). Moreover, when STM4538 was expressed from its own promoter selleck chemicals llc in the low-copy plasmid pMW118, the YK5009 strain showed an LDC-positive phenotype (Fig. 1a). However, the phenotype of the yfhK::Tn10dCm insertion was a false negative because this transposon insertion had no influence on LDC activity. We further compared the expression of a chromosomal cadA–lacZ fusion in strains JF3068 (wild-type), YK5007 (STM4538::Tn10dCm) and YK5011 (ΔSTM4538) using β-galactosidase assays. Following 30 min of acid stress, the level of cadA expression in the STM4538 mutants was approximately twofold lower than that in the wild-type (Fig. 1b). Together, these data suggest

that the PTS permease STM4538 is positively involved in the control of cadBA expression. To assess the potential role of STM4538 in the proteolytic activation of CadC, we performed an immunoblot PI3K Inhibitor Library analysis of total protein extracts from the S. Typhimurium wild-type and ΔSTM4538 strains harboring pACYC184-HA-CadC. N-terminally HA-tagged CadC (HA-CadC) was expressed under the control of its own promoter in the low-copy plasmid pACYC184. The cells were grown in E glucose medium to an OD600 nm of 0.6 and subjected to acid stress. As shown in Fig. 2, HA-CadC levels rapidly decreased in the wild-type background, as previously reported (Lee et al., 2008).

However, despite wild-type levels of cadC transcription (data not shown), HA-CadC levels were slightly increased in the ΔSTM4538 null mutant after acid stress, indicating impaired proteolytic processing filipin of CadC. These results suggest that the PTS permease STM4538

is required for the proteolytic activation of CadC signaling in S. Typhimurium. To gain further insight into the signaling mechanism of CadC, which undergoes rapid proteolytic cleavage in response to low pH and lysine signals (Lee et al., 2008), we examined whether both signals are required for this proteolytic event. Immunoblot analysis was conducted on total protein prepared from the YK5005 (cadA::lacZ ΔcadC) strain harboring pACYC184-HA-CadC. Cells were grown in E glucose medium to an OD600 nm of 0.6 and exposed to three different types of signals. The samples were collected at the indicated times and immunoblotted with anti-HA antibodies. As shown in Fig. 3(a), proteolysis of CadC occurs strictly in response to a pH shift regardless of the lysine signal. On the other hand, the lysine signal is insufficient on its own to stimulate proteolysis. To further confirm the concomitant effects of CadC proteolysis on cadBA transcription, the β-galactosidase activity from a cadA-lacZ transcription fusion was measured 30 min after each treatment. As expected, cadA transcription was induced only when cells respond to both low pH and lysine signals (Fig. 3b). These results suggest that proteolytic processing is a necessary but not sufficient step for CadC activation.