Journal: Nucleic Acids Research
Article Title: LysR-type regulator LrhA promotes CRISPR-Cas immunity in Escherichia coli
doi: 10.1093/nar/gkag204
Figure Lengend Snippet: Screen for new regulators modulating transcription of cas genes. ( A ) Schematic illustration of the CRISPR-Cas locus in E. coli . Repeats (diamonds) in the CRISPR array are interspersed with 12 unique spacer sequences (rectangles). The promoter of the cas operon (P cas ) is indicated by the black arrow. The P cas DNA probe for pull-down assay contains a transcription start site (TSS) (red letter), LeuO binding site I and II (LBS I/II), and H-NS binding site (HBS). For the pull-down proteomic experiment, a biotinylated probe (biotin-P cas DNA) was immobilized onto streptavidin beads, followed by incubation with extracts from E. coli MC4100 Δ hns (ZJUTCBB0015) or BW25113 Δ hns (ZJUTCBB0009) before SDS–PAGE electrophoresis. ( B ) A heatmap shows the number of peptides pulled down by P cas DNA probe. ( C ) SDS–PAGE gel electrophoresis of nontargeted biocytin (negative control, NT) or biotin-P cas DNA-binding proteins. NT or biotin-P cas samples were digested in gel with trypsin, and corresponding peptides were analyzed by mass spectrometry. An arrow denotes the band corresponding to LrhA (34.593 kDa). An asterisk marks the higher-abundance band in the MC4100 Δ hns sample (proteins listed in Supplementary Table S9). ( D ) Pull-down assay of physical interaction between P cas and LrhA with a Flag-tag (Flag-LrhA). The biotinylated probe (biotin-P cas DNA) was immobilized onto streptavidin beads and incubated with Flag-LrhA from cell extracts before (lane 3, input) and after (lane 2, P cas ) pull-down, followed by western blot assay with antibody against Flag-tag. Nontargeted biocytin (NT) was used as a negative control (lane 1). ( E ) (Upper) Effects of 10 predicted TFs and LeuO on the P cas activity in E. coli BW25113 Δ hns Δ leuO (EC73). (Lower) Effects of YdcI, SlyA, LrhA, and LeuO on the P cas activity in E. coli BW25113 Δ hns Δ leuO Δ lrhA (EC77). The expression of TFs was induced by adding 10 mM arabinose. The P cas activity was evaluated by GFP/OD 600 at 37°C, with the relative expression level of Δ hns Δ leuO carrying empty vector pSU19 (Vector) as 1 arbitrary unit. All bars represent the mean, and error bars denote standard deviation. Individual biological replicates are shown ( n = 4). Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test. **** P < .0001; ** P < .01 ( F ) Phylogenetic analysis of 9 HTH-type P cas -binding TFs from 5–8 different bacteria species.
Article Snippet: DNA-binding proteins and the total cell extracts were added with SDS sample loading buffer and boiled for 5 min at 95°C, then 20 μl of the sample was loaded onto a 12% Bio-Rad stain-free SDS–PAGE gel (Bio-Rad, 1610185) and blotted onto PVDF membranes using the semi-dry transfer apparatus (Genscript).
Techniques: CRISPR, Pull Down Assay, Binding Assay, Incubation, SDS Page, Electrophoresis, Nucleic Acid Electrophoresis, Negative Control, DNA Binding Assay, Mass Spectrometry, FLAG-tag, Western Blot, Activity Assay, Expressing, Plasmid Preparation, Standard Deviation, Bacteria
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