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. 2020 Jan 13;12(1):90. doi: 10.3390/v12010090

Figure 5.

Figure 5

cAMP receptor protein (CRP) positively regulates the transcription of gene cas3 to affect phage infection. (A) The CRP protein was pulled down by magnetic beads bonded to the probe. In the upper protein band, 8 potential regulators were identified, and in the lower protein band, 63 potential regulators were identified. (B) The β-gal activity of the cas3 promoter was measured by reporter plasmids in WT, Δcrp (EC61), or CΔcrp (EC71) cultured in LB media supplemented with 0, 0.2, or 0.8 mg/mL glucose. In WT, the presence of glucose led to an approximately 4 times decrease in cas3 promoter activity. (C) In the control group, the plasmids without the anti-vB_EcoS_SH2 spacer (pGEX) were transferred into WT (EC15), Δcrp (EC605), Δhns (EC95), and ΔhnsΔcrp (EC115). In the experimental group, the plasmids containing the anti-vB_EcoS_SH2 spacer (pGEX3) were transferred into WT (EC16), Δcrp (EC606), Δhns (EC96), and ΔhnsΔcrp (EC116). The phage titre of ΔhnsΔcrp was approximately 3 times of Δhns. (D) The plasmids overexpressing LeuO were transferred into Δcrp. In the control group, the plasmids without the anti-vB_EcoS_SH2 spacer (pGEX) were transferred into Δcrp (EC143). In the experimental group, the plasmids containing the anti-vB_EcoS_SH2 spacer (pGEX3) were transferred into Δcrp (EC146). The phage titre of pleuOΔcrp was almost 3 times of WT (pleuO). Phage titres were measured by lytic infection efficiency assay, as described in Materials and Methods. All the data were mean ± SEM of at least three replicates, and the p value (* p < 0.05) was analyzed by the t-test.