As determined by DNase I footprinting (Figure 2d), a purified His-CRP protein in the presence of 2 mM cAMP protected a single distinct region upstream of each target gene against DNase I digestion in a dose-dependent pattern. Taken together, CRP-cAMP stimulated ompC and ompF, while repressing ompX through the CRP-promoter DNA association in Y. pestis. No autoregulation of CRP Both lacZ fusion reporter (Figure 3a) and primer extension (Figure 3b) assays showed almost the same levels of crp expression in both WT and Δcrp; moreover, the footprinting analysis (Figure 3c) indicated no direct association
between His-CRP and crp promoter region in the presence 2 mM cAMP. Thus, no transcriptional auto-regulation of CRP could be detected in Y. pestis under the growth conditions used in this work. Figure 3 No autoregulation of CRP. a) Idasanutlin LacZ fusion reporter. A promoter-proximal region of crp was cloned into pRW50 and transformed into WT or Δcrp to determine their promoter activities, respectively. This figure shows the increased mean fold for the activity in Δcrp relative to WT. b) Primer extension. Primer extension assay was performed for crp using total RNAs from WT or Δcrp. On the selleck kinase inhibitor right side, DNA sequences are shown from the bottom (5′) to the top (3′), and the transcription start sites are underlined. c) DNase I footprinting. The labeled upstream DNA fragment of crp was incubated with 0, 5, 10, 15, and 20 pmol of purified His-CRP
in lanes 1 to 5, respectively, in the presence of 2 mM cAMP. No footprint region was detected. No regulatory interaction between OmpR and CRP As determined Dichloromethane dehalogenase by both primer
extension and lacZ fusion reporter assays, the ompR gene was expressed at almost the same level in both WT and Δcrp; likewise, no difference in the crp expression was observed between WT and ΔompR (Figure 4). Moreover, the footprinting analysis indicated no direct association between the His-CRP protein and the ompR promoter region or between the His-OmpR-P protein and the crp promoter region (Figure 4). Accordingly, under the growth conditions used in this work, OmpR had no regulatory effect on crp, and in turn, CRP did not regulate ompR. Figure 4 No regulatory interaction between OmpR and CRP. For RT-PCR and LacZ fusion experiments, we show the mean fold increase of the mRNA level (RT-PCR) or the detecting promoter activity (LacZ fusion) for crp or ompR in ΔompR or Δcrp relative to WT. For primer extension experiments, we show the primer extension product for crp or ompR in WT or Δcrp or ΔompR, and DNA sequences on the right side from the bottom (5′) to the top (3′); the transcription start sites are underlined. For DNase I footprinting experiments, the labeled DNA probe of crp or ompR was incubated with 0, 5, 10, 15, and 20 pmol of purified His-CRP (with addition of 2 mM cAMP) or His-OmpR (in the presence of 25 mM acetyl phosphate) in lanes 1 to 5, respectively. No footprint region was detected.