In the DNase I footprinting experiments, coding or noncoding strand (261 bp in length) containing the predicted CRP binding site was labeled with [γ-32P] at the 5′ end, then, incubated with increasing amounts of His-CRP; after partial digestion with DNase I, the resulting fragments were analyzed by denaturing gel electrophoresis. Radioactive species were detected by autoradiography. Primer extension learn more analysis For the primer extension assay [4], an oligonucleotide primer (Table 1) complementary to a portion of the RNA transcript of each gene was employed to synthesize cDNAs from the RNA templates. Electrophoresis

of primer extension products was performed with a 6% polyacrylamide/8M urea gel. The yield of each primer extension product would

indicate the mRNA expression level of the corresponding gene in each strain, and further could be employed to map the 5′ terminus of RNA transcript for each gene. Results The sycO, ypkA and yopJ genes constitute a single operon The RT-PCR assay indicated that the sycO, ypkA and yopJ genes (designated as pCD12, pCD13 and pCD14 in Y. pestis 91001 [19], respectively) were transcribed as a single primary RNA (Fig. 1), and thereby these three genes constituted a single operon in Y. pestis Microtus strain Tariquidar clinical trial 201. Figure 1 Transcriptional organization of the sycO-ypkA-yopJ operon. Arrows represent the length and direction of transcription of sycO, ypkA and yopJ on pCD1. The horizontal arrow depicts the putative primary RNA transcript. The arrowheads indicate the location of primer pair and the expected amplicons. SC79 molecular weight genomic DNA and cDNA generated by RT were used as the templates for PCR and RT-PCR, respectively. To ensure that there was no contamination of genomic DNA in the RT reactions, negative controls of RT-PCR were performed using ‘cDNA’ generated without reverse Fossariinae transcriptase as templates. Reactions containing

primer pairs without template were also included as blank controls. As expected, both negative and blank controls of RT-PCR gave no amplicon (data not shown). CRP greatly represses transcription of the sycO-ypkA-yopJ operon Our previous cDNA microarray analysis showed that the transcription of sycO, ypkA and yopJ was repressed by CRP [4]. Herein, the real-time RT-PCR assays confirmed that these three genes were up-regulated by more than 50 folds in the Δcrp mutant in relative to the WT strain (Fig. 2). Taken together, transcription of the sycO-ypkA-yopJ operon was under the negative control of CRP. Figure 2 CRP-dependent transcription of sycO, ypkA and yopJ. Shown was the mean log2 ratio (Δcrp versus WT) of mRNA level for each gene. CRP greatly represses promoter activity of sycO-ypkA-yopJ To test the action of CRP on the sycO-ypkA-yopJ promoter activity, we constructed the sycO::lacZ fusion promoter consisting of a 690 bp promoter-proximate region of sycO and promoterless lacZ, and then transformed into the WT and Δcrp, respectively.