A Motif Co-Occurrence Approach for Genome-Wide Prediction of Transcription-Factor-Binding Sites in Escherichia coli

Supplementary Materials to

"A Motif Co-Occurrence Approach for Genome-wide Prediction of Transcription Factor Binding Sites in E. Coli"

(Martha L. Bulyk, Abigail M. McGuire, Nobuhisa Masuda, George M. Church)

Detailed Methods

Primers Used to Create the Knockouts
Colony PCR Primers
Primers Used in Primer Extension Analysis
Primers Used in Quantitative Real-time PCRs (Taqman Assays)
Sequencing Primers
RNA Isolation and Purification
Primer Extension Analysis
Affymetrix mRNA Expression Analysis
Real-time RT-PCR Amplification
Real-time RT-PCR Negative Controls
Probability two randomly chosen noncoding bases in E. coli are separated by a given spacing

Primers Used to Create the Knockouts (go to top of page)

Names and sequences of primers used to create the binding site knockouts:

ArgRpdhR Ni:
GCACGTTGACGTACATTAAAAGCACGTTGACGTAGTGCTACATATTGAGAGGGGTTGAGG

ArgRpdhR No:
GCGGCCGCCCAGTATTTATAAGCAAAGTGGCTAAAGG

ArgRpdhR Ci:
CACTACGTCAACGTGCTTTTAATGTACGTCAACGTGCCAAAATTGGTAAGTGAATCGGTTCAATTCG

ArgRpdhR Co:
GGATCCGCCATAGGCTGCTCTGGAC

GalRytfQ Ni:
AATGGTTTCCGTTGTCTCACTATGAAAAATGCGGCTACGGT

GalRytfQ No:
GCGGCCGCCTTGCTGTGCGGAACCG

GalRytfQ Ci:
CAACGGAAACCATTAACTTGTGATTAACGTTTTATTTACTTTTTTGAAG

GalRytfQ Co:
GGATCCGTGGTCATATAGAGAGATTTGTCTTTCAC

LexArsR Ni:
AGTGTGTTAGAAGACTCATGTAAGTTAGGATGATGACTTTAGCTCTGAGGCATTTTCACTCT

LexArsR No:
GCGGCCGCGTGCGTTGGATCGGAAGAG

LexArsR Ci:
AGTCATCATCCTAACTTACATGAGTCTTCTAACACACTCACGTCCTTGCAATAGTTTCAGTAT

LexArsR Co:
GGATCCCCTGATTGTTGCAGGTAGTGTCT

MetJybdH Ni:
TATGTCCTTATGGCTTTATGTCCTTATGCCAACAATAAGGACAACACAACATGCCT

MetJybdH No:
GCGGCCGCCATTGAGGATAATCTCCGGTTCCA

MetJybdH Ci:
GGCATAAGGACATAAAGCCATAAGGACATAAAATACTAGCATCGTGTTATAGTGCCTTC

MetJybdH Co:
GGATCCCGGTGGTTGCAGTGCCATA

PhoByafL Ni:
CCACAAAATTAACGCATATAGACGCGTTAATTATGTAAAAACATCCGTTTATTAAATTACTCA

PhoByafL No:
GCGGCCGCGGAAGCGGTAAAGTATTATTGATAAGAAGC

PhoByafL Ci:
GCGTCTATATGCGTTAATTTTGTGGTAATGGTTTATTAAGTTTGTTCAGAAACGATCCG

PhoByafL Co:
GGATCCTTTATCAGGCCGCGTACC

PhoB2845 Ni:
GACGGCAAACAGACGGCATAACGACGCTTAACAATCATTTACTATTGCACTGTTAATT

PhoB2845 No:
GCGGCCGCTGAAGGCATCCCACAACCC

PhoB2845 Ci:
GCGTCGTTATGCCGTCTGTTTGCCGTCAGTGATTTTTATCACAAAGGAAATATGCCTGAG

PhoB2845 Co:
GGATCCCACTGCATATATCAACACGACGACA

Names and sequences of primers used to create the transcription factor knockouts:

ArgR Ni:
CACGCAATAACCTTCACACTCCAAATTTATAACCATAAGTCACCCGATATGGTGG

ArgR No:
GCTGACAAATGCGGCCGCGGTTTTTAACAGTAGTGCAAGCGC

ArgR Ci:
GTTATAAATTTGGAGTGTGAAGGTTATTGCGTGTAATCTCTGCCCCGTCGTTTC

ArgR Co:
CGCGGATCCCACACCACTTACGGATACGGT

FruR Ni:
CACGCAATAACCTTCACACTCCAAATTTATAACCACAATTGCCCCTTGCGTAA

FruR No:
GCTGACAAATGCGGCCGCTGAGCAGATCGAAAAGCAATTACACAAA

FruR Ci:
GTTATAAATTTGGAGTGTGAAGGTTATTGCGTGTAAGCCGCGAACAAAAATACGCG

FruR Co:
CGCGGATCCGTAAAAAACCCGATGTTTACGGCTAAT

PdhR Ni:
CACGCAATAACCTTCACACTCCAAATTTATAACCATGAGTTCCTGTCTTAAGCCAC

PdhR No:
GCTGACAAATGCGGCCGCGCGTGTGTAAGTTTGCAATTCCGTT

PdhR Ci:
GTTATAAATTTGGAGTGTGAAGGTTATTGCGTGTAGTGATTTTTCTGGTAAAAATTATCCAGAAGATGTTG

PdhR Co:
CGCAGATCTGAAGACTGGAAGGACGCCATAT

In all of the above No and Co primer sequences, the restriction sites are underlined. For the binding site knockout Ni and Ci primers, the binding site replacement sequences are underlined. A universal primer replacement sequence is underlined for the transcription factor knockout Ni and Ci primers.

Colony PCR Primers (go to top of page)

Names and sequences of primers used for identification of mutant using colony PCR:

ArgpdhR wt:
AATGAATTTAAATTCGTTTTAATTGAATTAAAAATCACA

ArgpdhR mut:
CACTACGTCAACGTGCTTTTAATGTACGTCAACGTGCCA

GalytfQ wt:
GATGAAAGCGATTACAAAC

GalytfQ mut:
GACAACGGAAACCATTAAC

LexarsR wt:
AACTGGATAATCATACAGTACATGCAGGTTATAAAACCAGCA

LexarsR mut:
AAAGTCATCATCCTAACTTACATGAGTCTTCTAACACACTCA

MetybdH wt:
TTTTATTTAGACATCTAAACGTCTTGATTGCC

MetybdH mut:
TTGGCATAAGGACATAAAGCCATAAGGACATA

PhoyafL wt:
TTAACTGAAATATATATGTTAATTTTATAATAA

PhoyafL mut:
TTAACGCGTCTATATGCGTTAATTTTGTGGTAA

Pho2845 wt:
GTTAATTACAGTTATCTTAATTGTTTAAAAAAA

Pho2845 mut:
GTTAAGCGTCGTTATGCCGTCTGTTTGCCGTCA

Primers Used in Primer Extension Analysis (go to top of page)

Names and sequences of primers used as probes in primer extension analysis:

23S 5'map:
GTTCATATCACCTTACCGACGCTTA

pdhR 5’map:
GGAGAGTGCCTTCGAGGATCAAAAAC

aceB 5’map:
AATTTGCTTCTCCTGCTCGCCATAC

argR 5’map:
GCAACGCGGCGACGATTT

Primers Used in Quantitative Real-time PCRs (Taqman Assays) (go to top of page)

Names and sequences of primers and fluorogenic probes used in Taqman assays:

pdhR-probe:
TTTTTGATCCTCGAAGGCACTCTCCGCC

aceB-probe:
ATTCTACCGCTTCGGCAGTAAGAATTTGCTTCTC

rrlH-probe:
CTGGGTTTCCCCATTCGGAAATCGCC

aroP-probe:
CTACCCAGGAATAACCCGGTCCCTATCGC

ppa-probe:
TCGATTTCGTATTTGATCGGATCTGCGTTAGC

yafL-probe:
TTTTCTTCATTTGCCAGCGCCACCACAT

ybdH-probe:
CACACCGCGCGAGAAAGTTGTTCATCAGT

ytfQ-probe:
CCGCTCCATTAACCGTTGGATTTTCGC

b2845-probe:
ACCACAGCCCCTGCCGAACCTAACTGA

ybdL-probe:
CTTCCACAACTTGGCACCACTATTTTCACCC

dinJ-probe:
CTTTTCACGCGCGACCTTTGTGAGG

b2844-probe:
AACGCCGGTACGTTCTATTAACGCTTTCAC

aroP-QRPf:
GATGGAAGGTCAACAGCACGGC

aroP-QRPr:
CCTGGCCCTGCGGACTGTATTA

ppa-QRPf:
ACTCAACGTCCCTGCGGGTAAAG

ppa-QRPr:
CGAACAGTGCGCCGCTCTCT

ytfQ-QRPf:
GTCTCGGCAGCCATGTCGTCT

ytfQ-QRPr:
CACTTTTCGCCACATTGGTTTCTG

dinJ-QRPf:
GCGCCCGAATCGATGAAGA

dinJ-QRPr:
CGCGTAAATCAAACGGCAATG

yafL-QRPf:
CTTTGTATTGTCGGGATTACTGTTGATTTG

yafL-QRPr:
TGCTTTTTCAGATGAGTTTGGTACTGTTTTA

b2844-QRPf:
GTTGTGATTGTCGGGGCGTTAC

b2844-QRPr:
CCAAGAATTACTTCATCCACCGCATA

b2845-QRPf:
CTAATATTTGGTCTAAAGAAGAAACTCTGTGGA

b2845-QRPr:
CCAGGCGACCAGAGCAGTAATAAA

ybdH-QRPf:
GGCCCGGCTAACTACTTTTCACATC

ybdH-QRPr:
AATGGCGCGTTTGCCGTAGAT

ybdL-QRPf:
AAATAACCCTCTGATTCCACAAAGCAA

ybdL-QRPr:
TGTAAATAGCGCGGACCATCAAAA

aceB-QRPf:
AGGCAACAACAACCGATGAACTG

aceB-QRPr:
ATGCGTCACCAGCTCAGTCAGAA

pdhR-QRPf:
CTCTCCGATGTGATTGAGCAGCA

pdhR-QRPr:
GGGACGGGAGACGTCAAACTGT

rrlH-QRPf:
GACTAAGCGTACACGGTGGATGC

rrlH-QRPr:
CCTATGGATTCAGTTAATGATAGTGTGTCG

Sequencing Primers (go to top of page)

aroP-bsKO
CTTTATGCATGGTTGAAGATGAGTTGC

pdhR-bsKO
TAGACTTTACGTAGGAAAGGAGTTTTTTAACGAT

dinJ-bsKO
GTATATTTATTCAGCTTGAATTGTAGCTCAAATGAG

yafL-bsKO
GCAAGGACATACTCAATAATGGCTGGTC

ppa-bsKO
TCTTCATTTTCCTTTTTCACCAGTTTTAAGAC

ytfQ-bsKO
AAACTATAGACAATTCGTTATGTAACAGATTGCG

ybdH-bsKOv2
GGTGATTGAAACTTCCTGGATGTGAAA

ybdL-bsKO
GGAATCAGAGGGTTATTTGTCATTTTTATTG

b2844-bsKO
CCGATAGGTGTCCGTAACGCCC

b2845-bsKO
TGGTGGGTTAGTGGTTGCAAACCTTAC

RNA Isolation and Purification (go to top of page)

Bacterial lysis and isolation of crude total RNA was achieved using hot acid phenol. Briefly, cultures were pelleted by centrifugation at 5000 g. Cell pellets were stored at -80°C. Cells were resuspended in acidic phenol:chloroform, 5:1 solution, pH 4.5 (Ambion; Austin, TX) pre-warmed at 65°C. RNA was extracted three times with acidic phenol:chloroform by addition of acidic phenol:chloroform, vortexing, incubating at 65°C for 3 min, incubating on ice for 3 min, then centrifuging for 5 min. The RNA was purified once with chloroform at room temperature, ethanol precipitated, and resuspended in DEPC-treated H2O. RNA samples were quantified at A260 and A280 using a spectrophotometer.

Primer Extension Analysis (go to top of page)

Primer extension analysis was performed as described before. Briefly, 10 mg total cellular RNA, [gamma-32P]-end-labeled gene-specific probe, and [gamma-32P]-end-labeled 23S-specific probe were heated at 65°C 90 min in hybridization buffer and then slowly cooled down to allow for specific annealing of the probes. The 23S-specific probe served as an internal quantitation control for each RNA sample. Afterwards, a mix of AMV reverse transcriptase, actinomycin D, and dNTPs in reverse transcription buffer was added at 42°C and the reaction was allowed to proceed for 1 hr. The reaction was then incubated with RNase I for 15 min at 37°C. The products were purified by phenol:chloroform extraction and ethanol precipitation, and then run on denaturing acrylamide gels. The gels were scanned on a Molecular Dynamics Storm PhosphorImager and quantified using ImageQuant™ software.

Affymetrix mRNA Expression Analysis (go to top of page)

Genome-wide mRNA expression analysis using Affymetrix™ GeneChip® oligonucleotide arrays was performed essentially as described previously. Briefly, to enrich for mRNA, reverse transcriptase and primers specific to 16S and 23S rRNA were used to synthesize cDNAs from total RNA. Then, rRNAs were removed by incubation with RNase H, which specifically digests rRNA within an RNA:DNA hybrid. The cDNAs were then removed by DNase I digestion, and the enriched mRNA was then purified on QIAGEN RNeasy columns. The purified, enriched mRNA was fragmented by heat and ion-mediated hydrolysis, and labeled at the 5’ ends with gamma-S-ATP using T4 polynucleotide kinase. The thiolated RNA was then labeled with PEO-iodoacetyl-biotin, and hybridized to the chip. After washing, the chip was stained with streptavidin, followed by staining with biotin-conjugated anti(streptavidin) antibody, and then finally by phycoerythrin-conjugated streptavidin.

Real-time RT-PCR Amplification (go to top of page)

RT-PCR reactions were carried out in iCycler IQ Real-Time Detection Systems (Bio-Rad, CA). SuperScript One-step RT-PCR with Platinum Taq kits (Invitrogen, CA) were used for all RT-PCR amplification in a total volume of 50 ml, which contained 200 ng total RNA, 5 mM MgSO4, 500 nM forward and reverse primers, and 200 nM fluorogenic probe. RT-PCR amplification for each RNA sample was performed in triplicate wells. One ‘no RT’ (without reverse transcriptase) control for each RNA sample and one ‘no RNA’ (substituted RNA with dH2O) control for each primer and probe set were also performed. The one-step RT-PCR condition is as following: 15 min at 50°C and 5 min at 95°C, followed by a total of 45 two-temperature cycles (15 sec at 95°C and 1 min at 60°C). Relative gene expression data analysis was carried out with the standard curve method.

Real-time RT-PCR Negative Controls (go to top of page)

ArgR bsKO in aroP-pdhR IGR:

ArgRpdhR #27:

yqeF: 0.84 +/- 0.16

yafL: 0.96 +/- 0.31

GalR bsKO in ppa-ytfQ IGR:

GalRytfQ #58:

yqeG: 1.2 +/- 0.39

ybdH: 1.2 +/- 0.15

PhoB bsKO in dinJ-yafL IGR:

PhoByafL #1 col.6:

ytfQ: 0.93 +/- 0.16

PhoB bsKO in dinJ-yafL IGR:

PhoByafL #2 col.1:

ybdL: 1.1 +/- 0.43

PhoB bsKO in yqeF-yqeG IGR:

PhoB2845 #3 col.2:

ppa: 2.1 +/- 2.0

PhoB bsKO in yqeF-yqeG IGR:

PhoB2845 #9 col.6:

aroP: 44 +/- 24

MetJ bsKO in ybdH-ybdL IGR:

MetJybdH #3 col.5:

pdhR: 3.0 +/- 0.99

MetJ bsKO in ybdH-ybdL IGR:

MetJybdH #8 col.1:

dinJ: 1.3 +/- 0.19

Probability two randomly chosen noncoding bases in E. coli are separated by a given spacing (go to top of page)

Probability p(x) that two randomly chosen, noncoding bases in the E. coli genome are separated by a spacing x.