Curation Information

Publication
Deep sequencing analyses expands the Pseudomonas aeruginosa AmpR regulon to include small RNA-mediated regulation of iron acquisition, heat shock and oxidative stress response.;Balasubramanian D, Kumari H, Jaric M, Fernandez M, Turner KH, Dove SL, Narasimhan G, Lory S, Mathee K;Nucleic acids research 2013 Oct 23; (): [24157832]
TF
AmpR [P24734, view regulon]
Reported TF sp.
Pseudomonas aeruginosa PAO1
Reported site sp.
Pseudomonas aeruginosa PAO1
Created by
Dinara Sagitova
Curation notes
-

Experimental Process

Transcription profiles of wild-type PAO1 and ampR mutant were compared in the presence and absence of sub-MIC b-lactam exposure by RNA-seq. ChIP-Seq analyses were performed using a 3x-V5-tagged AmpR. The functionality of the tagged AmpR in vivo was verified by determining the MIC and ChIP–qPCR studies. ChIP-Seq data was validated by performing ChIP-qPCR for selected targets using the V5-tagged AmpR strain.

ChIP assay conditions
For ChIP-Seq studies, the 3x-V5-tagged AmpR containing strain was grown and exposed to sub-MIC b-lactam stress.
ChIP notes
Protein–DNA interactions were then cross-linked in vivo with formaldehyde (final concentration of 1%) at room temperature for 20 min and quenched for 15 min with 0.25 M glycine. After three washes with 1× PBS, the cells were resuspended in 1 ml lysis buffer (10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% deoxycholic acid and 0.5% N-laurylsarcosine) containing a protease inhibitor cocktail (Roche). After chilling on ice, the cells were sonicated to shear the DNA to a size range of 0.5–1 kb. A 50 -µl aliquot of the supernatant was stored as the input DNA and the rest was immunoprecipitated using DynaBeads Protein G (Life Technologies), which was previously equilibrated and bound with anti-V5 monoclonal antibody (Sigma) as per manufacturer instructions. After immunoprecipitation overnight, the beads were washed five times with RIPA buffer (50 mM HEPES pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP40, 0.7% deoxycholic acid, 50 mM NaCl in 1× TE). The beads were resuspended in 100 µl elution buffer (50 mM Tris-HCl, 10 mM EDTA, 1% SDS) at 65°C for 30 min, centrifuged to remove residual beads and incubated at 65°C overnight to reverse the cross-link. TE buffer was then added (100 µl) and the samples were treated with RNase (37°C for 2 h). The immunoprecipitated proteins were removed with proteinase K (55°C for 2 h), and the DNA was cleaned using the Qiagen Mini Reaction Cleanup kit. RNase and proteinase K treatment was also performed for the input DNA. DNA concentrations were determined using Quant-iT PicoGreen dsDNA Kit (Life Technologies). Before proceeding further, AmpR occupancy of the ampC promoter was determined using qPCR as described previously (21). The DNA samples were then poly-A-tailed, blocked with biotinylated ddATP, purified and processed on the Helicos sequencer as described in the RNA-Seq section.

Transcription Factor Binding Sites


TTTTCTGGCAGGTT
TCGGCAGCCATTTT
TGTTATGGAAATGT
TTTGTTCAAAAAAT
TCTGCTTATGTATT
TCCACTGCAATATT
TGGGTGGCAAGTTT
GCATCTGAAAACTT
TTGGTTAATAGTTT
TCTGCTCCAAATTT
TGAGTTTCAATATT
TCATTCGCAAAGTT
TGGGCTCGATAATT
TCTCCTTTAGGCTT
GCTTTTGGAAGGTT
ACTTATTGAAATTA
TATTCGCCGAACTT
GGTGCCCGAAAGGT
TATTCGCCGAACTT
GGTGCCCGAAAGGT
CCTGCGCGAAAAGT
GTTTATCAGAACTT
CCGAATCCATATTT
TGGGCTTGAAAATG
TCTGTCCGAATCGT
TAGCCTTCGAACTT
TTTTCTGGCAGGTT
TCGGCAGCCATTTT
TGTTATGGAAATGT
TTTGTTCAAAAAAT
TCTGCTTATGTATT
TCCACTGCAATATT
TGGGTGGCAAGTTT
GCATCTGAAAACTT
TTGGTTAATAGTTT
TCTGCTCCAAATTT
TGAGTTTCAATATT
TCATTCGCAAAGTT
TGGGCTCGATAATT
TCTCCTTTAGGCTT
GCTTTTGGAAGGTT
ACTTATTGAAATTA
TATTCGCCGAACTT
GGTGCCCGAAAGGT
TATTCGCCGAACTT
GGTGCCCGAAAGGT
CCTGCGCGAAAAGT
GTTTATCAGAACTT
CCGAATCCATATTT
TGGGCTTGAAAATG
TCTGTCCGAATCGT
TAGCCTTCGAACTT
GCCAAAGACAATACGGAAACTCAGGGAATCCACCATGGATGGT None
CATCGAAGACCAAGTCCACCACTTGGCTCCATTCCAAGGTGAACAGGCTGCACTCCAGTCAGTACTGAGCCTTTTGGATGA None
TTTGTTACCGGCGT None
TACCCGTCAACCTGGTCAGGTCCGGAAGGAAGCAGCCACAG None
GAAGGTCCACGGCAACCTGACCTCCCTGGACGCCATCTACCACTTCGGTACCCCGGGCGTAGGTCTGCGTCCGTACGTGTCGGCTGGTCTGGCTCACCAGAACATCACCAACAT None
CCTCCATGCCCGTCGCGAC None
CGTCCCTTCCCCGATCCTTCGGGGTT None
AACCGTATCAACCTTGGGAGGGCCCGAGCGTATAAAAAAAGGCGCGAGGAAGGTGATTACTTCTTCATCGACACTTGAATCGCCTGCATAACCGTTATCTCCCCCGCGCCT None
TCCAGCTCGAGCTGCGCCTGGCGCGCCTCGGCGGCGGCCATGTGGGCCAGGTCAACGGCGCGCTCGCTGTCGCTGCGGTCGCGCCCCCAGAGATCGAGCTTGTAGTTCAGGCCGATCTCGGTGGAGTTGTTCCAGGAAGTGGTCCGCGCCAGGTCGCCGGGGCCGTAGAAATAGTCGTCCGGCCAGCGGTGGC None
ATGGTACGTACAGCAGCGTCCAGATCAGCAGCGGTCAGGTCAGCCTGCTTGGTCTTGGCGATCTCTTCCAGCTGGGCACGGGTAACGGTACCGACTTTCTGGGAGTTCGGACGAGCGGAGCCGCTGGTGATGCCAGCTGCTTTTTTCAGCAGGACGGCAGCCGGGGTGCTCTTGGTTTCGAAGGTGAAGCTACGGTCGCTGTAAACGGTGATGATCACAGGAGTCGGCAGGCCCGGCTCTTGGCCTTGGGTCTTGGCGTTGAACGCCTTGCAGAATTCCATGATGTTCACGCCGTGCTGACCCAGGGCAGGG None
CCTATTTGGTCTTGCTCCGAGTGGGGTTTACCTAGCCACGCACTGTTGCCAGCCGTGCGGTGCGCTCTTACCGCACCTTTTCACCCTTACCGGCGCTGTTGCGCTTAGGCGGTTATTTTCTGTGGCACTTTCCGTAGGCTCGCGCCTCCCAGGCGTTACCTGGCACTCTGCCCTATGGAGCCCGGACTTTCCTCCACCGCGCTATTGCGCGAC None
ACCCAGGAACGGCTGCCGTCCCCCACCGGATTTGAAGTCCGGCCACGCCACC None
CGGTGTGCATGTCCGCACGACGGAAAGCCTTAAGGTCTACTGCAACCGCCACCTTGAACTTTCGGGTTCAAGGGC None
GAAACGGCGGCGGGTTTTGTTGTGTGCGTGGGAAATGTTATTCCCGGTAACCGGACCCTTACCGGTTACTTGACAGACTCTAGACATGCCTCAGCCCTCTAT None
TATGGGGAGGGGGAAGTTCGGGGTTCAAGTCCGGACCGCTAG None
AAGCGCCAAAGACAATACGGAAACTCAGGGAATCCACCATGGATGG None
CAGCGAATTTACGAGTTCTGCTACCAGCTCGGCATGCCCCTCAAGTTTTGTTACCGGCG None
TTACCCGTCAACCTGGTCAGGTCCGGAAGGAAGCAGCCACAGCGGGA None
GGAGATTGCTGTTATGGAAATGTCCACCTTAAGGGGAACACGATGAACAACGTTCTGAAATTCTCTGCTCTGGCTCTGGCTGCTGTTCTGGCCACCGGTTGCAGCAGCCACTCCAAAGAAACCGAAGCTCGTCTGACCGCTACCGAAGACGCAGCTGC None
CGTCCCTTCCCCGATCCTTCGGGGTT None
ACCGTATCAACCTTGGGAGGGCCCGAGCGTATAAAAAAAGGCGCGAGGAAGGTGATTACTTCTTCATCGACACTTGAATCGCCTGCATAACCGTTATCTCCCCCGCGCCTCA None
TAGCCACGCACTGTTGCCAGCCGTGCGGTGCGCTCTTACCGCACCTTTTCACCCTTACCGGCGCTGTTGCGCTTAGGCGGTTATTTTCTGTGGCACTTTCCGTAGGCTCGCGCCTCCCAGGCGTTACCTGGCACTCTGCCCTATGGAGCCCGGACTTTCCTCCACCGCGCTATTGCGC None
CGACGAAGACAATAAAAACAACACGTAACGACTCCAGCACAACAAAAACAAAATCGCGGAGGCGCAGCTAACTGATTCTTTTGGAGAGGAGTTGCTGTCGGGACCCGTCCCGCAGCCAGTCGGAAGAAGAATAAAACTGCCTTGAGGCAGCGCACAGACTGGTTGGATCGCTCGACGATCATGGCAGCATCAGCGACCAAAGCAATCCGTTTGCTATTGAACTCCCAGCCTGGGAGATATCCCTG None
CCACCCAGGAACGGCTGCCGTCCCCCACCGGATTTGAAGTCCGGCCACGCCACC None
GCCAGTTGGACCGGTGTGCATGTCCGCACGACGGAAAGCCTTAAGGTCTACTGCAACCGCCACCTTGAACTTTCGGGTTCAAGGGC None
TATGGGGAGGGGGAAGTTCGGGGTTCAAGTCCGGACCGCTAGGGC None

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
TTTTCTGGCAGGTT
... ... rsmZ
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TCGGCAGCCATTTT
... ... rsmZ
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TGTTATGGAAATGT
... ... oprI
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TTTGTTCAAAAAAT
... ... oprI
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TCTGCTTATGTATT
... ... crcZ
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TCCACTGCAATATT
... ... PA3623 rpoS
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TGGGTGGCAAGTTT
... ... oprF
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
GCATCTGAAAACTT
... ... sigX oprF
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TTGGTTAATAGTTT
... ... lasR
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TCTGCTCCAAATTT
... ... ampR ampC PA4111
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TGAGTTTCAATATT
... ... PA4141 PA4142 PA4143 PA4144 PA4145
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TCATTCGCAAAGTT
... ... PA4141 PA4142 PA4143 PA4144 PA4145
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TGGGCTCGATAATT
... ... PA4141 PA4142 PA4143 PA4144 PA4145
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TCTCCTTTAGGCTT
... ... PA4141 PA4142 PA4143 PA4144 PA4145
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
GCTTTTGGAAGGTT
... ... PA4421 PA4420 ftsL ftsI murE murF mraY murD ftsW murG murC ddlB ftsQ ftsA ftsZ PA4406.1 lpxC rnpB
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
ACTTATTGAAATTA
... ... rsmZ
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TATTCGCCGAACTT
... ... ladS PA3973 PA3972 PA3971 amn thiD thiE hemL
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
GGTGCCCGAAAGGT
... ... ladS PA3973 PA3972 PA3971 amn thiD thiE hemL
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TATTCGCCGAACTT
... ... ladS PA3973 PA3972 PA3971 amn thiD thiE hemL
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
GGTGCCCGAAAGGT
... ... ladS PA3973 PA3972 PA3971 amn thiD thiE hemL
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
CCTGCGCGAAAAGT
... ... sigX oprF
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
GTTTATCAGAACTT
... ... oprF
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
CCGAATCCATATTT
... ... lasR
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TGGGCTTGAAAATG
... ... clpX lon hupB PA1804.1 ppiD
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TCTGTCCGAATCGT
... ... PA2763 PA2762 PA2764
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified
TAGCCTTCGAACTT
... ... PA4421 PA4420 ftsL ftsI murE murF mraY murD ftsW murG murC ddlB ftsQ ftsA ftsZ PA4406.1 lpxC rnpB
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details qPCR [quantitative real-time] (ECO:0005660) - Experimental technique details RNA-Seq (ECO:0005664) - not specified not specified