Wild-type enteroaggregative Escherichia coli. Arlette Darfeuille-Michaud. 17-2ÎlacZ. 17-2 deleted of the lacZ gene. This study. 17-2ÎlacZÎpcnB. 17-2ÎlacZ ...
Supplementary Table S1. Strains, plasmids and oligonucleotides used in this study. Strain
description, relevant genotype
source
17-2 17-2ΔlacZ 17-2ΔlacZΔpcnB 17-2ΔlacZΔpcnBΔfur 17-2ΔlacZΔpcnBΔaggR 17-2ΔlacZΔpcnBΔdam 17-2ΔlacZΔpcnBΔfurΔdam BW25113 BW25113 furΩkan BW25113 damΩkan MC4100 H1717
Wild-type enteroaggregative Escherichia coli 17-2 deleted of the lacZ gene 17-2ΔlacZ deleted of the pcnB gene 17-2ΔlacZΔpcnB deleted of the fur gene 17-2ΔlacZΔpcnB deleted of the aggR gene 17-2ΔlacZΔpcnB deleted of the dam gene 17-2ΔlacZΔpcnBΔfur deleted of the dam gene E. coli K12, ∆(araD-araB), ∆lacZ4787 BW25113 fur insertional mutant (KEIO collection) BW25113 dam insertional mutant (KEIO collection) F-, Δ(argF-lac)U169, araD139, rpsL150, relA1, deoC, rbs, flbB5301 FURTA-reporter strain, MC4100 aroB, fhuF::λlacMu
Arlette Darfeuille-Michaud This study This study This study This study This study This study Datsenko and Wanner, 2000 Baba et al., 2006 Baba et al., 2006 Laboratory collection Stojiljkovic and Hantke, 1994
lacZ transcriptional reporter fusion cloning vector, AmpR sci-1 promoter cloned into SmaI site of pGE593 sci-2 promoter cloned into SmaI site of pGE593 cirA promoter cloned into SmaI site of pGE593 Fur production vector, AmpR Suicide vector , Tn, KanR Recombination vector, phage λ recγβα operon under the control of the pBAD promoter, CmR Cloning vector, T7 promoter, AmpR sci-1 promoter cloned into pT7.5 sci-2 promoter cloned into pT7.5
Eraso and Weinstock, 1992 This study This study This study Tardat and Touati, 1993 Rossignol et al., 2001
Plasmids pGE593 psci1::lacZ psci2::lacZ pcir::lacZ pBT4-1 pNKBOR pKOBEG pT7-5 pT7-sci1 pT7-sci2
Chaveroche et al., 2000 Tabor and Richardson, 1985 This study This study
pT7-fur1 pT7-fur2 pT7-cir
fur1 box cloned into pT7.5 fur2 box cloned into pT7.5 cirA promoter cloned into pT7.5
This study This study This study
Oligonucleotides For gene deletiona ΔlacZ-5 GTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGATACAGCTATGTGTGTAGGCTGGAGCTGCTTCG ΔlacZ-3 TTTGTGTTTTTTAAATAGTACATAATGGATTTCCTTACGCGAAATACGGGCATATGAATATCCTCCTTAGTTC ΔpcnB-5 GCGTCATGCTGAGCTATGATTAGCCGCTATTTTTTTGTCCTGAATGATGTTGTGTAGGCTGGAGCTGCTTCG ΔpcnB-3 CTGCTCCAGCGGAGAGGCCAGATTGCTGCCTATGGCAATATACGCCACTGCATATGAATATCCTCCTTAGTTC Δfur-5 TGTCACTTCTCGTAATGAAGTGAACCGCTTAGTAACAGGACAGATTCCGCTGTGTAGGCTGGAGCTGCTTCG Δfur-3 CGTGTATAAAAAAGCCAACCCGCAGGTTGGCTTTTCTCGTTCCGGCTGGCCATATGAATATCCTCCTTAGTTCC ΔaggR-5 ACTCTAACGCAGAGTTGCCTGATAAAGACATTTTTTCATGTGAGAATGATTGTGTAGGCTGGAGCTGCTTCG ΔaggR-3 CATCACCAATATGTTTATAGCAATCTCAAATAATGATATAAAACATATTTCATATGAATATCCTCCTTAGTTCC Δdam-5 GCTGTCGGAGCTTTCTCCACAGCCGGAGAAGGTGTAATTAGTTAGTCAGCTGTGTAGGCTGGAGCTGCTTCG Δdam-3 CAGAATTGAGGGGGCAATCAAATACTGTTTCATCCGCTTCTCCTTGAGAACATATGAATATCCTCCTTAGTTC For promoter cloning into pGE593. Promsci1-5 Promsci1-3 Promsci2-5 Promsci2-3 Promcir-5 Promcir-3
CGCACCATGATCGTCTCTGTATCGC CTGAAACGAACTGCTCATGGCTCTCTC CTGGGGATTGGTGTTATGATCGGC CTCGTGGGGGGCGGGTTTTATC GGCAACCAGACGGTATACCTGTGGC GCGTACGGCAGCATCGCGGTG
For competition in gel shift experimentsb.
Fur-compet-up Fur-compet-bot RpoN-compet-up RpoN-compet-bot
TATGCCGGATAATGATAATCATTATCTATGCCG CGGCATAGATAATGATTATCATTATCCGGCATA TATGCCGAAGGGTGGCACGATGATTGCATATGCCG CGGCATATGCAATCATCGTGCCACCCTTCGGCATA
Sequencing oligonucleotides. for sequencing chromosomal insertion using pNKBOR. seq-NKBOR CAGAGATTTTGAGACACAACGTGGCTTTG for sequencing lacZ fusions. seq-lacZ-fus GTTGGGAAGGGCGATCGGTGCGG for sequencing pT7.5 insertions. seq-T7 GACAGCTTATCATCGATAAGCTTGGGCTGC For cloning into pT7.5 vector and gel shifts. using the double PCR techniquec Promsci1-pT7-5 CTCACTATAGGGAGACCGGAATTCGAGCTCACGGTGACAAGCGCACCATGATC Promsci1-pT7-3 GGTCGACTCTAGAGGATCCCCGGGCTGAAACGAACTGCTCATGGCTCTCTCC Promsci2-pT7-5 CTCACTATAGGGAGACCGGAATTCGAGCTCTGGGGATTGGTGTTATGATCGGCATC Promsci2-pT7-3 GGTCGACTCTAGAGGATCCCCGGGCTCGTGGGGGGCGGGTTTTATCAAG Promcir-pT7-5 CTCACTATAGGGAGACCGGAATTCGAGCTC GGCAACCAGACGGTATACCTGTGGCTG Promcir-pT7-3 GGTCGACTCTAGAGGATCCCCGGGCGTACGGCAGCATCGCGGTG using site-directed mutagenesisd, e Afur GCTCGAATTCCGGTCTCCCTATAGTGAGTCGTATTAATTTCG Bfur1 TAGGGAGACCGGAATTCGAGCTCAGTCCTGATTATTTGCATTATATCGCCCGGGGATCCTCTAGAGTCGACC Bfur2 TAGGGAGACCGGAATTCGAGCTCTCCTATAATGATCAAAATTAAATCAGCCCGGGGATCCTCTAGAGTCGACC
