As shown in Fig. 4, CRP protected two distinct DNA regions (sites 1 and 2) against DNase I digestion in a dose-dependent pattern. Only site 1 contained the CRP box-like sequence. Figure 4 DNase I footprinting assay. The labeled DNA probe was incubated with various amounts of purified His-CRP (lanes 1, 2, 3, 4, and 5 contained 0, 500, 1000, 2000 and 3000 ng, respectively), and subjected to DNase I footprinting assay. Lanes G, A, T and C represented the Sanger sequencing reactions. On the right-hand

side was indicated the protected regions (bold line). The DNA sequences of footprints were shown from the top (3′) to the bottom (5′). The transcription start site of sycO was determined by primer extension assay. A single primer extension product was detected and thus a single click here CRP-dependent Selleck ARRY-438162 promoter was transcribed for sycO-ypkA-yopJ (Fig. 5). Compared to the WT,

a much stronger primer extension product was detected in the Δcrp. Since the yield of primer extension product would indicate the mRNA 4EGI-1 molecular weight expression level of sycO in each strain, data presented here confirmed the repression of sycO-ypkA-yopJ by CRP. Figure 5 Primer extension analysis. Electrophoresis of the primer extension products was performed with a 6% polyacrylamide/8M urea gel. Lanes C, T, A and G represented the Sanger sequencing reactions. The transcriptional start sites were underlined. The primer extension results could be also employed to map the 5′ terminus of RNA transcript for sycO (i.e. the transcription start site of sycO-ypkA-yopJ) (Fig. 6). The -10 and -35 core promoter elements were predicted accordingly. Figure 6 Structural organization of the sycO-ypkA-yopJ promoter region. The sycO-ypkA-yopJ promoter-proximate sequences (100

bp upstream to 50 bp downstream the start codon of sycO) from Y. pestis Antiqua (biovar Antiqua), KIM5 (Mediaevalis), CO92 (Orientalis) and 91001(Microtus), as well as those from Y. pseudotuberculosis IP32953 and Y. enterocolitica Demeclocycline 8081, were aligned and conserved nucleotide sites were labeled with asterisks. The CRP binding sites were underlined, and the invert repeats in the CPR box was showed with two invert arrows. Showed also were transcriptional/transcriptional start sites and promoter -10 and/or -35 elements. The determination of CRP-binding sites, transcription start site, and core promoter element (-10 and -35 regions) promoted us to depict the structural organization of CRP-dependent promoter, giving a map of CRP-promoter DNA interaction for sycO-ypkA-yopJ (Fig. 6). Discussion CRP and the sycO-ypkA-yopJ operon CRP specifically bound to the sycO promoter-proximate region and directly repressed the expression of sycO-ypkA-yopJ in Y. pestis biovar Microtus strain 201. The sycO-ypkA-yopJ promoter-proximate regions were extremely conserved in Y.