Curation Information

Publication
Genome-scale analyses of Escherichia coli and Salmonella enterica AraC reveal noncanonical targets and an expanded core regulon.;Stringer AM, Currenti S, Bonocora RP, Baranowski C, Petrone BL, Palumbo MJ, Reilly AA, Zhang Z, Erill I, Wade JT;Journal of bacteriology 2014 Feb; 196(3):660-71 [24272778]
TF
AraC [P0A9E0, view regulon]
Reported TF sp.
Escherichia coli str. K-12 substr. MG1655
Reported site sp.
Escherichia coli str. K-12 substr. MG1655
Created by
Erill Lab
Curation notes
-
External databases
ArrayExpress (A-MEXP-2346) http://www.ebi.ac.uk/arrayexpress/arrays/A-MEXP-2346/. ArrayExpress (E-MTAB-1916) http://www.ebi.ac.uk/arrayexpress/arrays/E-MTAB-1916/.

Experimental Process

ChIP-chip was performed on cells growing on LB or LB+arabinose. Chip results were validated with ChIP-qPCR. Motif discovery was performed in the ChIP enriched (ratio >mean + 2*stdev) regions and a motif matching the known consensus was identified. A DNA array was also used to analyze expression patterns on w-t and AraC mutants in presence or absence of arabinose. Expression changes >4 fold between w-t and mutant upon arabinose were deemed significant. The conservation of identified sites was evaluated with multiple sequence alignments against several related species. Site directed mutagenesis and ChIP-PCR was performed on the putative ytfQ AraC-binding site, and its regulation was further established with Beta-gal assays.

ChIP assay conditions
40 ml E. coli cells expressing C-terminally TAP-tagged AraC (for ChIP-chip; SAC003; Table 1), or C-terminally FLAG-tagged AraC (for ChIP/qPCR; AMD187; Table 1) were grown in LB or LB + 0.2% arabinose at 37 °C to an OD600 of 0.6-0.8.
ChIP notes
Cells were crosslinked for 20 minutes with formaldehyde (1% final concentration), pelleted by centrifugation and washed once with Tris-buffered saline (TBS). Cell pellets were resuspended in 1 ml FA lysis buffer (50 mM Hepes-KOH, pH 7, 150 mM NaCl, 1 mM EDTA, 1% Triton X- 100, 0.1% sodium deoxycholate, 0.1% SDS) with 2 mg/ml lysozyme and incubated at 37 °C for 30 minutes. Samples were then chilled and sonicated for 30 minutes in a Bioruptor sonicator (Diagenode) with 30 s on/30 s off pulsing at maximum amplitude. Samples were pelleted in a microcentrifuge to remove debris and supernatants (“chromatin”) were saved, 1 ml FA lysis buffer was added, and samples were stored indefinitely at -20 °C. For each immunoprecipitation (IP), 500 μl chromatin was incubated with 300 μl FA lysis buffer, 20 μl Protein A Sepharose slurry (50%) in TBS and either 1 μl anti-β (RNA polymerase subunit) antibody (NeoClone) or 2 μl M2 anti-FLAG antibody (Sigma) for 90 minutes at room temperature with gentle mixing on a Labquake Rotisserie Rotator (Thermo Scientific). For ChIP of AraC-TAP, Protein A sepharose and antibody was replaced with IgG sepharose. Beads were then pelleted at 1,500 x g in a microcentrifuge for 1 minute. The supernatant was removed and the beads were resuspended in 750 μl FA lysis buffer and transferred to a Spin-X column (Corning). Beads were then incubated for 3 minutes with gentle mixing on a rotisserie rotator before being pelleted at 1,500 x g in a microcentrifuge for 1 minute. Equivalent washes were performed with FA lysis buffer, high salt FA lysis buffer (50 mM Hepes-KOH, pH 7, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), ChIP wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet-P40, 0.5% sodium deoxycholate) and TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). After the TE wash, beads were transferred to a fresh Spin-X column and eluted with 100 μl ChIP elution buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS) for 10 minutes at 65 °C with occasional agitation. Eluted samples were centrifuged at 1,500 x g in a microcentrifuge for 1 minute. Supernatants were decrosslinked by boiling for 10 minutes and cleaned up using a PCR purification kit (Qiagen). For all ChIP/qPCR and ChIP-chip experiments, 20 μl chromatin was decrosslinked by boiling for 10 minutes and cleaned up using a PCR purification kit (Qiagen). This sample served as the “input” control. ChIP samples from 8 independent cultures grown in the presence of arabinose were combined and ethanol precipitated to reduce volume. 8 samples were required to generate sufficient DNA for dye labeling. These samples were labeled with Cy3 dye as described previously (23). 100 ng input DNA was labeled with Cy5 dye. Dye-labeled samples were combined and hybridised to the custom-designed Agilent microarrays in a hybridisation oven (Agilent) according to the manufacturer’s instructions. Microarray slides were washed and scanned using an Agilent scanner and Agilent software was used to determine Cy3/Cy5 ratios for all probes. Any probes with a score of <1,000 pixels for either Cy3 or Cy5 were removed as these are likely to indicate regions deleted in the strain used. We selected an arbitrary cut-off for Cy3/Cy5 ratio of two standard deviations above the mean to select putative AraC-bound regions.

Transcription Factor Binding Sites


CAGAGCAGGAAAATCCATA
CACAGCAGATTAATCCATA
TATAGCAGGAAGATCCATA
CGCAGCAATTTAATCCATA
TTCAGCAGGATAATGAATA
CATAGCATTTTTATCCATA
CACGGCAGAAAAGTCCACA
TTTAGCGGGAAAAGACATA
CAGAGCAGGAAAATCCATA
CACAGCAGATTAATCCATA
TATAGCAGGAAGATCCATA
CGCAGCAATTTAATCCATA
TTCAGCAGGATAATGAATA
CATAGCATTTTTATCCATA
CACGGCAGAAAAGTCCACA
TTTAGCGGGAAAAGACATA

Quantitative data format: Fluorescence ratio of ChIP-chip between cells grown with and wihout arabinose. Range: 0.009 to 7.435

CAGAGCAGGAAAATCCATA 1.866737438
CACAGCAGATTAATCCATA 5.316664004
TATAGCAGGAAGATCCATA 0.455512073
CGCAGCAATTTAATCCATA 0.627087798
TTCAGCAGGATAATGAATA 1.254519369
CATAGCATTTTTATCCATA None
CACGGCAGAAAAGTCCACA 7.435441595
TTTAGCGGGAAAAGACATA None
TGCAACGTCCGCGACGAATATCCTGCGGGCGGGTGGTACCGGAAAGCAGAGCTGGCACCCA 0.261716722
ACCACCAATGTAGCGGGAGTAGTCAACGTTGCCCGGCGCGTGGCACAAACGGGTAATCTCT 0.511521009
GCGCTGCGCCCGGAGTTTGCCGAAAACCCGAACGCGATGTTCGTATTGTGGAAAGACCACA 0.269564754
TCAATCGGTGCGGGCGTCGAGCCGGTACTGTCAACGCCAATCCCGACCACAGCTGCGCGCT 0.537268214
GTTCCGTCATCATCCGCGTGACTACATTGAGTCAATGGAAGCGGCACTGAAAACCGTGCTT 1.188902704
AAAATTGCCCTTTCTGCCAACGGGGATACCACTCTACGCTGGTGGCGATCTCTTCACCGGT 3.100785641
GATGGAGTGAAACGATGGCGATTGCAATTGGCCTCGATTTTGGCAGTGATTCTGTGCGAGC 2.167463113
AAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAA 6.586735319
GTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCAC 7.435441595
GGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTGGGTTAGCGAGAAG 5.062785509
TTCATACTCCCACCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTC 3.093493944
AATGATCCCCTGCTGCCGGGATACTCGTTTAACGCCCATCTGGTGGCGGGTTTAACGCCGA 0.625449503
ACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCGATAAATCTGCTTGAGCAATT 0.474197432
CGCCAATCATCGCACTCTTTTCCAGCCGCTACTCACTCAAACATATCTTGTTGTTTCTGGT 0.35263943
TGCATAATACGAGATCATATGCCCGGCGGCAGGAATCGAAATTCCTACGTTATGCGCCAGC 0.508616705
AAAAAAGTCATTTTATCTTTGGCTCTGGGCACGTTTGGTTTGGGGATGGCCGAATTTGGCA 0.787523643
CCAGAACGGCTACCAATCACCGTAGCCAATAAAGTGATAGCTTGCAGGATAACGACCAGCA 1.164419749
GCAAACTGGAAAGTACGTTTGCAGTGAAATAACTATTCAGCAGGATAATGAATACAGAGGG 1.254519369
TTTGGAATTAAGTACAATAAGAAGAGGAACATTTATGAAGTCTGCATTAAAGAAAAGTGTC 0.429581912
GGTGGCGGAAAATATGGATCTTCCTGCTATACCGCCACGCTATCGCAGCAGTTATTTCGCC 0.455512073
ATCTCATACCCTTTGTTGAACAGGCTGGCATTACGCATTTTCTGTTGCAGTTCGTCAGGCA 0.450627983
CGAACGTCAGGGCCAGGAACTGTATAAAGAGATGCAGAAACGTGGCTGGGATGTCAAAGAA 0.323395706
TCATCACCAGCGGAACGGTATCCATTGGCTTACCTTTGGCGTTAACAAACTGGTCATCCAC 0.260564804
TCGTTATTTGTACTCCGGACCCCAAACTCGGCTCTGCCATCGTCGCGAAAGCGCGTGGCTA 0.354291804
AGCCAGGCTGTCGATCGCGTTCAATGTTTTTTCGCCATCCGGCACGGCAATCTTAATAACC 0.649635591
CTGGTGAAGCAACCGGAAGAGCCGTGGTTCCAGACCGAATGGAAGTTTGCCGATAAAGCCG 0.313544184
TCCGCCATAGCGGATTGTGACATAACGGCTGCCAGACCAATGGCTGCCAGGGCTTTAGTAA 1.518691884
TTTCCGATTAATTTAACGAATGTCATTCGTTTTTGCCCTACACAAAACGACACTAAAGCTG 2.532731019
CTAAAAGATAAGTGACTGTGTTGACATAGTTTTAGCGAGAAATTAATTCTCCATAGGAGAG 2.110215054
CTAATCTTATGGATTAATCTGCTGTGCATTCGACAATTTGTCTGACAAATTGGCTTTCCCT 5.316664004
TTTGGTACCCATGCGGGATGTCTTCTTTTTAACCAGTCAATAGGCCGCATTACCTGGCGTT 4.430793777
CAGTGATAATTAATGACGAAATGAATGACGTGCATTTTCCACATCTTTGAGTTGCGGTTAT 3.99588682
CAGACGTTATTCTTATTTCAGATCATCGTCAGAATTGACTCCACGATCACATTTCGGACCG 0.777877644
TTACGCATCCAATCTCTACCTTCACCTGAGTAACTGGTGTTCTGAACAGAGTCTGAACGGC 0.284297487
CCGACACTCTTCATAAAGTTAAACATGCGCATCATTTGGGTCACATTACTCTGTGCCTGGG 0.275575135
TTAATCGCCGCTCGTGTGGTGCTGGGCATTGCTGTCGGGATCGCGTCTTACACCGCTCCTC 0.5414033
GCTACCGAAACAAACATATTCATACGCCGCGTATCCCGCAAAGAACGTGGCGTTAAAGCAG 1.947671953
CTGCGTGAGTTGTTCACGTATTTTTTCACTATGTCTTACTCTCTGCTGGCAGGAAAAAATG 1.703384968
ATAAATATGGATTAAATTGCTGCGACATGTCGTTATGTGATGGATATTCCAATTTTCAAAT 0.627087798
TTATGACCCTGCCGCATGGCAGGGTTTTTTATACCTGTAGATCATCATAATCCATATCATG 1.121094302
CTATCGCATTAACATTAATGTTGTGTTTAGCCCATTCGTTCGCCATCAATCGCGTCACACC 0.319502194
TTATCAATATCGCGTCAATGCTCTCCTTCCAGGGCGGGATCCGTGTGCCTTCTTATACCGC 0.240262003
TGCCTTGTCGGGTTTAGGATTAGCGATAACAATGATTCGACATTCAGCACTTTTGTTTTTT 0.242577355
TTAGGTGAATAAATTGGGTTTTTCTTGCATCGCTGAGCCGTTGACTCGATTGCCGTAGCTC 0.243872906
GTGGCAATAGAATAAGGGTGTCTGTTAATCGCATTGACGCCAAAATAACTTAATGTCATAC 0.243954132
TCAGAATGGTCTAAGGCAGGTCTGAATGAATACCCAGTATAATTCCAGTTATATATTTTCG 0.267855491
TGTTGGAATTGTCAGGCAGTTAAGGCAATTATCATTTTTTACAATGCGTTTACGTAATGTC 0.260845679
TGTTTTGTAGGTAAGGTGTTATGTTGCCTTGTCGTACCATTATCAACACGATAATAATTAA 0.28668976
TAAGGGTTTTTTTATGCCCGCGATAAATAAACACACCTTATTCCACCACCGCCTGCAAGCG 0.284650492
CAGGGTTATTACGCCAGAAGAACTTATTTATTCTTTGCGCGCTCGAAGGAGGCAACGATTT 0.240539313
CTTCGAGCACTACAAAGACCTCGAAAAAGGCAAGTGGGTGAAAGTTGAAGGTTGGGAAAAC 0.457815413
TTCGCATCTTCACCGGCTTCGTCGGTCATTTTCAGAACGCCAACCGGACGGCAACGGATCA 0.360058916
CATCAACCACACCCTGTCTCTGGACGGTGACCCGGTTGACGTACTGGTCCCAACTCCGTAC 0.347805104
AGAACATCGCGGTGGACATGAAGCGGTCAACGAACAGTGCGCCGCTCTCTTTGTCGATTTC 0.325926197
ACGTCCCTGCGGGTAAAGATCTGCCGGAAGACATCTACGTTGTTATTGAGATCCCGGCTAA 1.905006105
TTCCTTTAAAAATATGTGGCAAACAAGTGCCGAGTATTATAGCCAACTCGCGCCGAATGTC 1.305717518
GGCTACGGTTATGGATTTTCCTGCTCTGTATACCGTCTTAAAACTGGTGAAAAAGGAAAAT 1.866737438
TTGGCCGCTCCATTAACCGTTGGATTTTCGCAGGTCGGATCGGAATCAGGCTGGCGTGCCG 1.900964709
TTCCTGCTTTTGCTGACCATCGGCAATTTTCAACGTGATTCCGCGCTTTTCGGCTTCACTT 1.172400178
GTTCCTTCGTTGCACAAGGGGTGGATGCGATCTTTATCGCTCCGGTGGTCGCGACAGGTTG 0.490177736
CACTGCCGACAACATCCTCGAAGGCAAGTTGATTGGTGACTGGCTGGTAAAAGAAGTGAAT 0.402696642
TCGGCAAAGCCTTTCTTACGGTCAATGGCGACGCTGGCCCCAACGGTGCCCTGCAGCTCCA 0.367457447
AATATCAAAATCATCCGCTCGCAGTCAGGTGACTTCACCCGCAGTAAAGGCAAAGAAGTCA 0.265090676

Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.

Site sequence Regulated genes Gene diagram Experimental techniques TF function TF type
CAGAGCAGGAAAATCCATA ytfQ,
... ... ytfQ ppa ytfR ytfT yjfF
Experimental technique details Beta-gal reporter assay - Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - Experimental technique details Site directed mutagenesis (ECO:0005667) - repressor monomer
CACAGCAGATTAATCCATA araF, araG, araH,
... ... araF araG araH ftnB
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - activator monomer
TATAGCAGGAAGATCCATA
... ... dcp ydfG ydfH
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - not specified monomer
CGCAGCAATTTAATCCATA araE, ygeA
... ... araE ygeA
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - activator monomer
TTCAGCAGGATAATGAATA araJ
... ... araJ
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - activator monomer
CATAGCATTTTTATCCATA araB, araA,
... ... araB araA araC yabI
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - activator dimer
CACGGCAGAAAAGTCCACA araB, araA,
... ... araB araA araC yabI
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - activator dimer
TTTAGCGGGAAAAGACATA araF, araG, araH,
... ... araF araG araH ftnB
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - activator monomer