β-galactosidase activity

was measured for evaluating the sycO-ypkA-yopJ promoter activity in each strain. Since the crp mutation had an effect on the copy number of recombinant or empty pRS551 plasmid [4], a normalized fold change in the activity of each fusion promoter in WT in relative to Δcrp was calculated to avoid the influence of copy number of pRS551 (Table 2). Table 2 Promoter activity determined with the sycO:lacZ reporter see more fusion   Fold change (Δcrp/WT) Normalized fold change of promoter activity in Δcrp in relative to WT LacZ fusion Plasmid copy number Miller units   PsycO-lacZ 0.006 0.182 30.33 β-Galactosidase activity (miller units) was detected as the promoter activity. An extremely low promoter activity was detected for the Δcrp or WT transformed with empty pRS551 (data not shown). Copy number of recombinant pRS551 (PsycO-lacZ) was determined by real-time quantitative PCR, the detecting fold change of plasmid copy number was set to be 1 to generate a normalization factor that was subsequently used for generating the normalized fold change of promoter activity

(miller units) in the Δcrp in relative to the WT. Each experiment was done in learn more triplicate. Accordingly, the β-galactosidase activity in the Δcrp increased compared to the WT when they grew in the ‘TMH-1mM cAMP’ medium, indicating that CRP greatly repressed the promoter activity of sycO-ypkA-yopJ (Table 2). CRP binds to promoter-proximate SCH772984 ic50 Protirelin region of sycO-ypkA-yopJ A CRP box-like sequence was found in the promoter-proximate region of sycO-ypkA-yopJ [4], indicating the direct association of CRP with the sycO-ypkA-yopJ promoter region. Further EMSA experiments showed that the cAMP-CRP complex bound to the sycO-ypkA-yopJ promoter region in a CRP dose-dependent manner (Fig. 3a). CRP could not bind to the target DNA in the absence of cAMP. To validate the specifiCity of CRP-DNA interaction, YPO0180 and YPO1099 [gene IDs in CO92 [20]] were used as negative controls

(Fig. 3b). The PCR-generated upstream DNA of YPO0180 did not harbor the predicted CRP binding site, while the YPO1099 upstream region gave an extremely low score value of 0.96 during the pattern matching analysis using the CRP consensus (sycO gave a score value of 8.57) [4]. Both of them gave negative EMSA result, even the CRP protein was increased to 4 μg in a single reaction mixture (Fig. 3b). Figure 3 Electrophoretic mobility shift assay. The band of DNA fragment containing the promoter region of sycO disappeared with increasing amounts of CRP protein, and a retarded DNA band with decreased mobility turned up (Fig. 3a), which presumably represented the CRP-DNA complex. But for YPO0180 and YPO1099, the CRP-DNA complex did not appear even His-CRP was increased to 4 μg for each reaction mixture (Fig. 3b). Therefore, CRP specifically bound to the sycO-ypkA-yopJ promoter region and directly repressed the transcription of sycO-ypkA-yopJ.