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TRANSCRIPT
SUPPLEMENTAL INFORMATION
Dual function of the McaS small RNA in controlling biofilm formation
Mikkel Girke Jørgensen1,4, Maureen K. Thomason2,3,4, Johannes Havelund1, Poul Valentin-Hansen1,5 and Gisela Storz2,5
1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
2Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development,
Bethesda, MD USA.
3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC USA.
4Co-first authors
5Co-corresponding authors Email [email protected] or [email protected]
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Table of Contents
Supplemental Figure S1 Additional truncations of pgaA 5’ UTR. Related to Figure 1. 4
Supplemental Figure S2 Evidence supporting conservation of McaS and CsrA binding sites. Related to Figure 2. 5
Supplemental Figure S3 Evidence supporting the dual functionality of McaS mutants, which are defective in 6
CsrA binding but are still able to regulate targets by base pairing. Related to Figure 2.
Supplemental Figure S4 Evidence supporting the conclusion that lower expression of McaS mutants is not the 7
primary reason for failure to regulate pgaA-lacZ. Related to Figure 2.
Supplemental Figure S5 Evidence supporting the specificity of CsrA for McaS and not other sRNAs such as the 9
RyhB sRNA. Related to Figure 3.
Supplemental Figure S6 Evidence showing that McaS regulates other CsrA targets such as glgC-lacZ. Related to 10
Figure 5.
Supplemental Figure S7 Quantitation of McaS, CsrB and CsrC levels throughout growth. Related to Figure 7. 11
Supplemental Figure S8 Effects of mutations in non-conserved GGA sequences. Related to Figure 2 and Discussion. 12
Supplemental Figure S9 Evidence supporting the dual binding of CsrA and Hfq to McaS. Related to Figure 7 14
and Discussion.
Supplemental Table S1 List of strains and plasmids used in this study 16
Supplemental Table S2 List of oligonucleotides used in this study 20
2
Supplemental Methods Detailed descriptions of strain and plasmid construction. 30
Supplemental References 33
3
Supplemental Figure S1. Assays of PM1205 ΔabgR-ydaL derivatives with (A) pgaA155-lacZ and (B) pgaA67-lacZ fusions transformed
with the control vector, pBR-McaS and plasmids expressing the McaS-2 and McaS-3 mutant derivatives. β-galactosidase activities of
the fusions were assayed with either 1 mM IPTG (black bars) or no IPTG (white bars). The average values from three independent
assays are shown and error bars are standard deviations of those values.
4
Supplemental Figure S2. Conservation of McaS. Alignment of McaS sequences across closely related species to include
representative strains of Escherichia, Shigella, Enterobacter, Citrobacter and Cronobacter. Residues conserved across all species are
indicated by an *. Potential CsrA GGA binding sites are highlighted in red.
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Supplemental Figure S3. Effects of wild-type and mutant McaS on (A) csgD-lacZ and (B) flhD-lacZ expression. Reporter strains
PM1205 ∆abgR-ydaL csgD-lacZ and PM1205 ∆abgR-ydaL flhD-lacZ were transformed with the control vector, pBR-McaS and
plasmids expressing the McaS-8 and McaS-9 mutant derivatives. β-galactosidase activities for the fusions were assayed as in
Supplemental Fig. S1.
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Supplemental Figure S4. (A) and (B) Levels of plasmid-expressed wild-type and mutant McaS in NM525 ΔabgR-ydaL (A) and
PM1205 ∆abgR-ydaL pgaA-lacZ (B). Overnight cultures were diluted to an OD600 of ~0.05 in LB and allowed to grow for 1.5 h at
37˚C upon which expression of the wild-type and mutant derivatives was induced with 1 mM IPTG for all samples in (A) and for
pBR-McaS-2, pBR-McaS-8 and pBR-McaS-11 in (B) or 70 µM IPTG for pBR-McaS, pBR-McaS-3 and pBR-McaS-10 in (B). Total
RNA was extracted and 10 μg was separated on an 8% polyacrylamide-7M urea gel, transferred to a membrane and probed with 32P-
labelled McaS specific oligonucleotide or 5S oligonucleotide as a control. (C) Effects of uniform wild-type and mutant McaS RNA
levels on pgaA-lacZ expression. β-galactosidase activities for the samples in (B) were assayed as in Supplemental Fig. S1. Mfold
(http://mfold.rna.albany.edu/?q=mfold) predicts multiple possible structures for most of the McaS mutants, many of which are similar
to structures predicted for the wild-type RNA.
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Supplemental Figure S5. RyhB RNA does not co-purify with CsrA. Strain SØ928 csrA-Flag was grown in LB medium to an OD600
of 1.0. At this point the culture was split and the chelator 2,2′-dipyridyl was added to one culture to induce RyhB synthesis. After 10
min, samples were harvested, cell extracts were prepared and incubated with anti-Flag M2-agarose beads. The beads were collected on
a small column, the filtrate was collected (unbound fraction; UB), and the beads were washed with IP buffer (wash fraction, W).
Subsequently, the proteins trapped on the beads were eluted with 1M arginine buffer (bound fraction; B). Total RNA was extracted
from the three fractions and examined for the presence of CsrB and RyhB RNA by Northern blot analysis.
9
Supplemental Figure S6. McaS activation of glgC-lacZ expression. The reporter strain PM1205 ∆abgR-ydaL glgC-lacZ was
transformed with the control vector, pBR-McaS, plasmids expressing McaS mutant derivatives, and pBR-CsrB. β-galactosidase
activity was assayed as in Supplemental Fig. S1.
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Supplemental Figure S7. Quantification of CsrB, CsrC and McaS RNA levels for wild-type MG1655 samples shown in Fig. 7. The
signal for each band was converted to the concentration of the specific sRNA species by comparison with in vitro transcribed sRNA
run on the same gel as described (Overgaard et al. 2009). To facilitate the quantification, the in vitro transcribed sRNAs were labeled
with tritium (-3H-CTP) during their synthesis.
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Supplemental Figure S8. (A) The sequences of the first 80 nucleotides of wild-type McaS and mutant derivatives. Nucleotides altered
for each mutant are boxed. Putative CsrA binding sites are shaded in red. (B) Effects of mutant derivatives on pgaA-lacZ expression.
The reporter strain PM1205 ΔabgR-ydaL pgaA234- lacZ was transformed with the control vector, pBR-McaS and plasmids expressing
McaS mutant derivatives indicated in (A). β-galactosidase activity was assayed as in Supplemental Fig. S1. Mfold
(http://mfold.rna.albany.edu/?q=mfold) predicts multiple possible structures for most of the McaS mutants, many of which are similar
to structures predicted for the wild-type RNA.
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Supplemental Figure S9. (A) CsrA and Hfq can simultaneously bind McaS and do not cooperate in binding. Samples containing a 5´
end-labeled McaS RNA (0.5 nM) were incubated with increasing amounts of Hfq protein (at the indicated concentrations) in the
absence and presence of CsrA protein (5 nM) and examined by gel mobility shift assays. (B) CsrA does not interact with Hfq-bound
RprA RNA. Samples containing a 5´ end-labeled RprA RNA (0.5 nM) were incubated with increasing amounts of CsrA protein (at the
indicated concentrations) in the presence of Hfq protein (10 nM) and assayed as in (B).
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Supplemental Table S1. Strains and plasmids used in this study.
NameMPK number
Genotype Source
MG1655 E. coli F- λ- ilvG- rfb-50 rph-1 Lab stock
BW25113 lacIq rrnBT14 ∆acZWJ16 hsdR514 ∆araBADAH33 ∆rhaBADLD78
(Datsenko and Wanner 2000)
TOP10 F- mcrA ∆(mrr-hsdRMS-mcrBC) φ80lacZ∆M15 ∆lacX74 nupG recA1 araD139 ∆(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-
Invitrogen
NM500 MG1655 mini-λ::tet N. Majdalani
TR1-5 MPK0336 E. coli K-12 TR1-5 csrA::kan Romeo et al 1993
MG1655-csrA::3xFLAG MG1655 csrA::3xFLAG::KanR This study
MG1655 ∆pgaA MG1655 ∆pgaA::kan This study
RH90 MC4100 rpoS359::Tn10 (Lange and Hengge-Aronis 1991)
MG1655 ∆rpoS MG1655 rpoS359::Tn10 This study
MG1655 ∆pgaA ∆rpoS MG1655 ∆pgaA, rpoS359::Tn10 This study
MG1655 ∆mcaS MG1655 ∆mcaS::kan This study
MG1655 ∆rpoS ∆mcaS MG1655 rpoS359::Tn10, ∆mcaS This study
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MG1655 ∆pgaA ∆rpoS ∆mcaS
MG1655 ∆pgaA, rpoS359::Tn10, ∆mcaS This study
PM1205 MPK0122 MG1655 mal::lacIq, ∆araBAD araC+, lacI'::PBAD-cat-sacB-lacZ, mini λ tetR
(Mandin and Gottesman 2009)
GSO613 MC4100 Δhfq::cat-sacB (Zhang et al. 2013)
GSO143 MPK0385 EH288 ∆csrB::kan (Hobbs et al. 2010)
GSO560 MPK0125 PM1205 lacI'::PBAD-csgD-lacZ, ∆abgR-ydaL::kan (Thomason et al. 2012)
GSO564 MPK0210 PM1205 lacI'::PBAD-flhD-lacZ, ∆abgR-ydaL::kan (Thomason et al. 2012)
GSO568 MPK0228 NRD686 PM1205 lacI'::PBAD-pgaA234-lacZ, ∆abgR-ydaL::kan (Thomason et al. 2012)
GSO569 MPK0106 MG1655 ∆abgR-ydaL::kan (Thomason et al. 2012)
GSO552 MPK0131 NM525 ∆abgR-ydaL::kan (Thomason et al. 2012)
GSO632 MPK0387 MG1655 TR1-5 csrA::kan This study
GSO633 MPK0386 MG6155 ∆csrB::kan This study
GSO634 MPK0281 PM1205 lacI'::PBAD-pgaA155-lacZ This study
GSO635 MPK0285b PM1205 lacI'::PBAD-pgaA155-lacZ, ∆abgR-ydaL::kan This study
GSO636 MPK0299 PM1205 lacI'::PBAD-pgaA108-lacZ This study
GSO637 MPK0303 PM1205 lacI'::PBAD-pgaA108-lacZ, ∆abgR-ydaL::kan This study
GSO638 MPK0300 PM1205 lacI'::PBAD-pgaA67-lacZ This study
GSO639 MPK0304 PM1205 lacI'::PBAD-pgaA67-lacZ, ∆abgR-ydaL::kan This study
GSO640 MPK0335 PM1205 lacI'::PBAD-pgaA30-lacZ This study
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GSO641 MPK0337 PM1205 lacI'::PBAD-pgaA30-lacZ, ∆abgR-ydaL::kan This study
GSO642 MPK0333 NRD686 PM1205 lacI'::PBAD-pgaA234-lacZ, ∆abgR-ydaL This study
GSO643 MPK0376 NM500 ∆pgaA::cat This study
GSO644 MPK0377 NRD686 PM1205 lacI'::PBAD-pgaA234-lacZ, ∆abgR-ydaL, ∆pgaA::cat
This study
GSO645 MPK0382 NRD686 PM1205 lacI'::PBAD-pgaA234-lacZ, ∆abgR-ydaL, ∆pgaA::cat, TR1-5 csrA::kan
This study
GSO646 MPK0350 PM1205 lacI'::PBAD-ydaM-lacZ This study
GSO647 MPK0353 PM1205 lacI'::PBAD-ydaM-lacZ, ∆abgR-ydaL::kan This study
GSO648 MPK0356 PM1205 lacI'::PBAD-ydeH-lacZ This study
GSO649 MPK0360 PM1205 lacI'::PBAD-ydeH-lacZ, ∆abgR-ydaL::kan This study
GSO650 MPK0359 PM1205 lacI'::PBAD-ycdT-lacZ This study
GSO651 MPK0363 PM1205 lacI'::PBAD-ycdT-lacZ, ∆abgR-ydaL::kan This study
GSO652 MPK0283 PM1205 lacI'::PBAD-glgC-lacZ This study
GSO653 MPK0287 PM1205 lacI'::PBAD-glgC-lacZ, ∆abgR-ydaL::kan This study
GSO654 MPK0322 MG1655 mcaS::cat-sacB This study
GSO655 MPK0326 MG1655 mcaS::cat-sacB, mini-λ::tet This study
GSO656 MPK0383 MG1655 mcaS::mcaS-3 This study
GSO657 MPK0384 MG1655 mcaS::mcaS-8 This study
GSO658 MPK0401a NM525 ΔabgR::ydaL::kan, ΔpgaA::cat This study
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Plasmid Relevant features Source
pKD4 (KmR); Km cassette flanked by FRT sites for recombineering; template plasmid (Datsenko and Wanner 2000)
pKD3 (CmR); Cm cassette flanked by FRT sites for recombineering; template plasmid (Datsenko and Wanner 2000)
pKD46 (AmpR); mini λ Red recominbase expression plasmid for recomineering (Datsenko and Wanner 2000)
pCP20 (AmpR); (CmR); pSC101repts, contains FLP recombinase (Cherepanov and Wackernagel 1995)
pNDM-220 (AmpR); mini R1, lacIq pA1/O4/O3 (Gotfredsen and Gerdes 1998)
pNDM-McaS pNDM-220; pA1/O4::mcaS (Jørgensen et al. 2012)
pNDM-McaSmut3 pNDM-220; pA1/O4::mcaSmut3 This study
pNDM-McaSmut8 pNDM-220; pA1/O4::mcaSmut8 This study
pSUB11 (AmpR); R6KoriV, template plasmid for 3x FLAG construction linked to Kan cassette
(Uzzau et al. 2001)
pBAD33 (CmR); p15; araC pBAD (Guzman et al. 1995)
pBAD-csrA3xFLAG pBAD33; pBAD::csrA3xFLAG This study
pBRplac (AmpR); Plac promoter based expression vector with AatII at transcription start site
(Guillier and Gottesman 2006)
pBR-McaS (AmpR); pBRplac with mcaS cloned into AatII and HindIII (Thomason et al. 2012)
pBR-McaS-2 mutant A14U G15C A16U mcaS cloned into pBRplac (Thomason et al. 2012)
pBR-McaS-3 mutant A33U C34G G35A mcaS cloned into pBRplac (Thomason et al. 2012)
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pBR-McaS-4 mutant G70C G71C A72U mcaS cloned into pBRplac (Thomason et al. 2012)
pBR-McaS-8 mutant A14U mcaS cloned into pBRplac This study
pBR-McaS-9 mutant A33U C34G G35A A39U mcaS cloned into pBRplac This study
pBR-McaS-10 mutant A14U A33U C34G G35A mcaS cloned into pBRplac This study
pBR-McaS-11 mutant A39U mcaS cloned into pBRplac This study
pBR-McaS-12 mutant A14U A33U C34G G35A G70C G71C A72T mcaS cloned into pBRplac
This study
pBR-McaS-13 mutant A14U G24T G25C A26T A33U C34G G35A mcaS cloned into pBRplac
This study
pBR-CsrB (AmpR); pBRplac with csrB cloned into AatII and EcoRI (Mandin and Gottesman 2010)
pBR-GcvB (AmpR); pBRplac with gcvB cloned into AatII and EcoRI (Mandin and Gottesman 2010)
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Supplemental Table S2. Oligonucleotides used in this study.
Number Name Sequence Use
McaS_NB McaS probe TCCGCGTCTTAAATCCGGCATTGTCTCCTCTGCGCCGGT
McaS Northern probe
RprA_NB RprA probe GCCCATCGTGGGAGATGGGCAAAGACTACACACAG RprA Northern probe
CsrB_NB CsrB probe CATCCTGGTGTGTCCTGCAGAAGTGTCATCATCCTG CsrB Northern probe
GcvB_NB GcvB probe GCAATTAGGCGGTGCTACATTAATCACTATGGACAG GcvB Northern probe
OmrA NB OmrA probe GTGCAAGAGACAGGGTACGAAGAGCGTACCGA OmrA Northern probe
CsrC NB CsrC probe TCCTGAGTCATTGTTCCTGTTAGCGTCCTCG CsrC Northern probe
MK0063 McaS probe AGCAGTGCATCCGCGTCTTAAATCC McaS Northern probe
MK0018 McaS probe CCAGACTCTACAGTACACACAGCAG McaS mutants Northern probe
5S 5S probe CGGCGCTACGGCGTTTCACTTCTG 5S Northern probe
5S_NB 5S probe CTACGGCGTTTCACTTCTGAGTTCCCGTATGTAGCATCACCTTC
5S Northern probe
MK0318 CsrB probe CTGCGTCATCCTCTTCGCTTCATCCAGAAGC CsrB Northern probe
MK0475 CsrC probe GACCCTCATCCTGAGTCATTGTTCCTG CsrC Northern probe
For in vitro transcription of various RNAs
T7_mcaS McaS primer with T7 site
GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGAGACAA
In vitro transcription forward primer for McaS
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T7_mcaS2 McaS-2 primer with T7 site
GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGTCTCAATGCCGGATTTAAGACGCGGATGCACTGCTGTG
In vitro transcription forward primer for McaS-2
T7_mcaS3 McaS-3 primer with T7 site
GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGAGACAATGCCGGATTTAAGTGACGGATGCACTGCTGTG
In vitro transcription forward primer for McaS-3
T7_mcaS8 McaS-8 primer with T7 site
GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGTGACAATGCCGGATTTAAGACGCGGATGCACTGCTGTG
In vitro transcription forward primer for McaS-8
T7_mcaS9 McaS-9 primer with T7 site
GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGAGACAATGCCGGATTTAAGTGACGGTTGCACTGCTGTGTGTAC
In vitro transcription forward primer for McaS-9
T7_mcaS10 McaS-10 primer with T7 site
GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGTGACAATGCCGGATTTAAGTGACGGATGCACTGCTGTG
In vitro transcription forward primer for McaS-10
T7_mcaS11 McaS-11 primer with T7 site
GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGAGACAATGCCGGATTTAAGACGCGGTTGCACTGCTGTGTGTAC
In vitro transcription forward primer for McaS-11
Bam_mcaS_R
Common McaS Rev primer
ccccGGATCCAAAAAATAGAGTCTGTCGACATCCGCCAGACTCTACAGTAC
Common McaS reverse primer for in vitro transcription and cloning into pNDM-220
T7_RprA RprA primer with T7 site
GAAATTAATACGACTCACTATAGGACGGTTATAAATCAACATATTGATTTATA
In vitro transcription forward primer for RprA
RprA_R RprA rev primer AAAAAAAGCCCATCGTGGGAGATG RprA reverse primer for in vitro transcription
T7-GcvB GcvB primer with T7 site
GAAATTAATACGACTCACTATAGGACTTCCTGAGCCGGAACGAAAAG
In vitro transcription forward primer for GcvB
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GcvB_R GcvB rev primer AAAAAAGGTAGCTTTGCTACCATGGTC GcvB reverse primer for in vitro transcription
T7_CsrB CsrB primer with T7 site
GAAATTAATACGACTCACTATAGGaGTCGACAGGGAGTCAGACAACGAAGTGAAC
In vitro transcription forward primer for CsrB
CsrB_R CsrB rev primer AATAAAAAAAGGGAGCACTGTATTCACAGCGCTCCCGGT
CsrB reverse primer for in vitro transcription
T7_CsrC_F CsrC primer with T7 site
GAAATTAATACGACTCACTATAGGATAGAGCGAGGACGCTAACAGGAACAATGA
In vitro transcription forward primer for CsrC
T7_CsrC_R CsrC rev primer AAGAAAAAAGGCGACAGATTACTCTGTCGCCTTTTTTCCTGACTC
CsrC reverse primer for in vitro transcription
For cloning McaS (and mutant versions) and CsrA into plasmids
pA1/O4_mcaS3
pNDM-McaSmut3 fwd primer
GCCTGACGTCGGCAAAAAGAGTGTTGACTTGTGAGCGGATAACAATGATACTTAGATTCACCGGCGCAGAGGAGACAATGCCGGATTTAAGTGACGGATGCACTGCTGTG
Forward primer for McaS-3 cloning into pNDM-220
pA/O4_mcaS8
pNDM-McaSmut8 fwd primer
GCCTGACGTCGGCAAAAAGAGTGTTGACTTGTGAGCGGATAACAATGATACTTAGATTCACCGGCGCAGAGGTG
Forward primer for McaS-8 cloning into pNDM-220
Pst_SD_csrA
pBAD33 cloning CsrA fwd
CCCCCTGCAGTCGATTAAGGAGGTCACATATGCTGATTCTGACTCGTCGAGT
Forward primer for amplifying CsrA3FLAG for cloning into pBAD33
Hind_FLAG_R
pBAD33 cloning CsrA rev
GGGGCTGCAGTTACTATTTATCGTCGTCTCTTTG Reverse primer for amplifying CsrA3FLAG for cloning into pBAD33
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MK0099 pBRplac fwd primer GCGACACGGAAATGTTGAATAC Forward primer for PCR and sequencing check for cloning into pBRplac
MK0100 pBRplac rev primer CAGTACCGGCATAACCAAGC Reverse primer for PCR and sequencing check for cloning into pBRplac
MK0098 McaS-pBR-fwd-aatII TATACTATGACGTCTCACCGGCGCAGAGGAGACAATG
Common forward primer for PCR of McaS, 5' AatII restriction site for cloning, use in PCR #3 for mutant construction with PCR #1, #2 as templates
MK0065 McaS-rev-hindIII GACCGGAAGCTTGAATGCGGCTATCTGCAAAG Common reverse primer for PCR of McaS, 3' HindIII restriction site for cloning, use in PCR #3 for mutant construction with PCR #1, #2 as templates
MK0096 McaS rev PCR #1 GTTCTCAGCCTGTATCAGTCT Common reverse primer for McaS mutant cloning PCR #1
MK0064 McaS fwd PCR #2 GCCACTGAATTCGAAATCTGTCACTGAAGAAAAT Common forward primer for McaS mutant cloning PCR #2
MK0443 McaS-8, McaS-10, McaS-12, McaS-13 fwd aatII
tatactatgacgtcACCGGCGCAGAGGTGACAATGCCGGATTTAAG
Forward primer for McaS-8, McaS-10, McaS-12, and McaS-13 mutants, aatII site for cloning, use with MK0065 for mutant construction
MK0444 McaS-9 fwd PCR #1 CCGGATTTAAGTGACGGTTGCACTGCTGTGTG Forward primer for McaS-9 mutant PCR #1; use with MK0096
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MK0445 McaS-9 rev PCR #2 CACACAGCAGTGCAACCGTCACTTAAATCCGG Reverse primer for McaS-9 mutant PCR #2, 5' end complimentary to MK0444; use with MK0064
MK0243 McaS-3, McaS-10, McaS-12 fwd PCR #1
CCGGATTTAAGTGACGGATGCACTG Forward primer for McaS-3, McaS-10, and McaS-12 mutants PCR #1; use with MK0096
MK0244 McaS-3, McaS-10 rev PCR #2
CTTAAATCCGGCATTGTCTCC Reverse primer for McaS-3, and McaS-10 mutant PCR #2, 5' end complimentary to MK0243; use with MK0064
MK0465 McaS-11 fwd PCR #1 CCGGATTTAAGACGCGGTTGCACTGCTGTGTG Forward primer for McaS-11 mutant PCR #1; use with MK0096
MK0466 McaS-11 rev PCR #2 CACACAGCAGTGCAACCGCGTCTTAAATCCGG Reverse primer for McaS-11 mutant PCR #2; 5' end complimentary to MK0465; use with MK0064
MK0483 McaS-12 rev PCR #2 GTCGACAAGGGCCAGACTCTACAGTACACACAGCAGTGCATCCGTCACTTAAATCC
Reverse primer for McaS-12 mutant PCR #2; 5’ end complimentary to MK0243; use with MK0064
MK0484 McaS-13 fwd PCR #1 CAATGCCTCTTTTAAGTGACGGATGCACTGC Forward primer for McaS-13 mutant PCR #1; use with MK0096
MK0485 McaS-13 rev PCR #2 GCAGTGCATCCGTCACTTAAAAGAGGCATTG Reverse primer for McaS-13 mutant PCR #2; 5’ end complimentary to MK0484; use with MK0064
Primers to create gene knockouts
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pgaA_del_F pgaA::kan knockout fwd
AAAAATCCGAAATCATGCATCGGAATTTACTGATTTAATTATTTTAATCCtgtgtaggctggagctgcttc
Forward primer for pgaA knockout PCR using pKD4 as template
pgaA_del_R pgaA::kan knockout rev GCGGCAGCAAGTTGATTATTACGTAATGCCTGCACGTATTCTGTGGGATAcatatgaatatcctccttag
Reverse primer for pgaA knockout PCR using pKD4 as template
MK0237 pgaA::cat knockout fwd ATACAGAGAGAGATTTTGGCAATACATGGAGTAATACAGGgtgtaggctggagctgcttc
Forward primer for pgaA knockout PCR using pKD3 as template
MK0464 pgaA::cat knockout rev ATCAGGAGATATTTATTTCCATTACGTAACATATTTATCCcatatgaatatcctccttag
Reverse primer for pgaA knockout PCR using pKD3 as template
MK0239 pgaA check fwd TCCGAAATCATGCATCGGAATTTAC Forward primer for pgaA knockout PCR check and sequencing
MK0240 pgaA check rev CACGGTTGCTCGGCGAGTAA Reverse primer for pgaA knockout PCR check and sequencing
csrA3FLAG_F
csrA FLAG tag fwd GGAAGTTTCTGTTCACCGTGAAGAGATCTACCAGCGTATCCAGGCTGAAAAATCCCAGCAGTCCAGTTACGACTACAAAGACCATGACGGTGATTATAAA
Forward primer used for chromosomal csrA-FLAG tagged amplification
csrA3FLAG_R
csrA FLAG tag rev AGTAAAGCGAAAAGACAATGGAGTGAAAGAGAAATTTTGAGGGTGCGTCTCACCGATAAAGATGAGACGCCATATGAATATCCTCCTTAG
Reverse primer used for chromosomal csrA-FLAG tagged amplification
Primers to create lacZ fusions
MK0174 PBAD-fwd CGACGAATTCGCGCTTCAGCCATACTTTTCATAC General forward primer for checking PBAD-lacZ fusion integration and sequence
MK0175 Deeplac rev CGGGCCTCTTCGCTA General reverse primer for
25
checking PBAD-lacZ fusion integration and sequence
MK0242 pgaA-lacZ rev TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACCGGGCACCTTTTTCTGCT
Common reverse primer for pgaA-lacZ fusion generation
MK0314 pgaA155-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATGACACTCTGCTCATCATTTC
Forward primer for pgaA155-lacZ truncation fusion generation; use with MK0242
MK0395 pgaA108-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATTCTCTTCCGCGTTTAATAAC
Forward primer for pgaA108-lacZ truncation fusion generation; use with MK0242
MK0396 pgaA67-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATTCTTTCTTTTCAGTTACCTG
Forward primer for pgaA67-lacZ truncation fusion generation; use with MK0242
MK0436 pgaA30-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATAGATTTTGGCAATACATGGA
Forward primer for pgaA30-lacZ truncation fusion generation; use with MK0242
MK0448 ydaM-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATGAATTATCTGATCATATGAC
Forward primer for ydaM-lacZ fusion generation; use with MK0450
MK0450 ydaM-lacZ Rev TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGTCCAGGGTATTGAAGTTGT
Reverse primer for ydaM-lacZ fusion generation; use with MK0448
MK0451 ycdT-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATAAAGGGATCTACAACCTACA
Forward primer for ycdT-lacZ fusion generation; use with MK0452
MK0452 ycdT-lacZ rev TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAA Reverse primer for ycdT-lacZ fusion generation; use with
26
CGACACTACTAATTCTCAAAT MK0451
MK0453 ydeH-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATGCACAAGGAACTGTGAAAA
Forward primer for ydeH-lacZ fusion generation; use with MK0454
MK0454 ydeH-lacZ rev TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCATCAATTTCCGTTG
Reverse primer for ydeH-lacZ fusion generation; use with MK0453
MK0315 glgC-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATTGCTCAACCTTTAAGCACGG
Forward primer for glgC-lacZ fusion generation; use with MK0317
MK0317 glgC-lacZ rev TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACTAAGTGATCGTTCTTCTCTA
Reverse primer for glgC-lacZ fusion generation; use with MK0315
Primers to integrate McaS-3, McaS-8 onto the chromosome
MK0202 McaS-cat-sacB fwd CTGTCACTGAAGAAAATTGGCAACTAAAGGTTAAAACCGTatcggcaatttcttttgcgttt
Forward primer for amplification of cat-sacB with region of mcaS homology for integration; use with MK0203
MK0203 McaS-cat-sacB rev TCTTAAATCCGGCATTGTCTCCTCTGCGCCGGTGACT Reverse primer for amplification
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GTaaaatgagacgttgatcggcacg of cat-sacB with region of mcaS homology for integration; use with MK0202
MK0204 McaS cat-sacB PCR check fwd
CCCGCAATTAAGAGCGCGAT Forward primer to PCR check integration of cat-sacB and mcaS-3, mcaS-8 chromosomal mutations
MK0205 McaS cat-sacB PCR check rev
GAATGCGGCTATCTGCAAAG Reverse primer to PCR check integration of mcaS-3, mcaS-8 chromosomal mutations
MK0468 McaS-3 chrom fwd PCR #1
GCAGTGCATCCGTCACTTAAATCCGGCAT Forward primer for McaS-3 integration on chromosome; use with MK0160 in PCR #1
MK0160 McaS chrom rev common primer PCR #1
GAAACTATCCGCGTAAGCGTGGC Common reverse primer for mcaS integration on chromosome; use with MK0468 in PCR #1 for mcaS-3, use with MK0472 in PCR #1 for mcaS-8
MK0467 McaS-3 chrom fwd PCR #2
ACCGGCGCAGAGGAGACAATGCCGGATTTAAGTGACGGATGCACTGCTGTGTGTACTGTA
Forward primer for mcaS-3 integration on chromosome; use with MK0159 in PCR #2
MK0159 McaS chrom rev common primer PCR #2
TCACGTCGCCAGTGCGATAAT Common reverse primer for McaS integration on chromosome; use with MK0467 in PCR #2 for mcaS-3, use with MK0471 in PCR #2 for mcaS-8
MK0472 McaS-8 chrom fwd PCR #1
CGTCTTAAATCCGGCATTGTCACCTCTGCGCCGGT Forward primer for mcaS-8 integration on chromosome; use with MK0160 in PCR #1
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MK0471 McaS-8 chrom fwd PCR #2
ACCGGCGCAGAGGTGACAATGCCGGATTTAAGACG Forward primer for mcaS-8 integration on chromosome; use with MK0159 in PCR #2
MK0469 McaS chrom common fwd PCR #3
CCTGAAGAAATGGGCTGCGATCCCTTGCAC Forward primer for mcaS integration on chromosome; use with MK0470 for PCR #3 for both mcaS-3 and mcaS-8 integration on chromosome
MK0470 McaS chrom common rev PCR #3
TGGATGCCAGTGGACATCGGTAGC Reverse primer for mcaS integration on chromosome; use with MK0469 for PCR #3 for both mcaS-3 and mcaS-8 integration on chromosome
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Supplemental Methods
Strain construction
(i) The MG1655-csrA::3xFLAG strain was constructed by amplifying the csrA gene using primers csrA_3FLAG_F and
csrA_3FLAG_R and plasmid pSUB11 as a template followed by recombineering into the csrA locus of strain BW25113/pKD46. The
csrA::3xFLAG::kan allele was moved into MG1655 by P1 transduction.
(ii) The MG1655 ∆pgaA strain was constructed by amplifying the flanking regions of pgaA using primers pgaA_del_F and
pgaA_del_R and plasmid pKD4 as a template followed by recombineering into strain BW25113/pKD46. Subsequently, MG1655
pgaA was generated by P1 transduction and excision of the kanR resistance cassette with plasmid pCP20 (Cherepanov and
Wackernagel 1995).
(iii) The MG1655 ∆pgaA::cat strain was constructed by amplifying the flanking regions of pgaA using primers MK0237 and MK0464
and the plasmid pKD3 as a template. The PCR product was recombineered into strain NM500 containing the chromosomal mini λ
cassette. The resulting ∆pgaA::cat mutation was transduced into the relevant strains by P1 transduction.
(iv) The MG1655 ∆rpoS and MG1655 ∆pgaA ∆rpoS strains were constructed by P1 transduction of the rpoS359::Tn10 allele from
strain RH90 into MG1655 and MG1655 ∆pgaA.
(v) The PM1205 ydaM-lacZ fusion strain was constructed as previously described (Mandin and Gottesman 2009). However, there is
no published transcription start site for ydaM, as a result, the 5’ end was determined to reside ~75 nt upstream of the start codon,
according to transcription start site mapping of the E. coli genome (Thomason et al unpublished results).
30
(vi) The McaS-3 and McaS-8 chromosomal mutants were constructed by first inserting a cat-sacB cassette at the mcaS chromosomal
locus of MG1655. The cat-sacB cassette was amplified from strain MC4100 Δhfq::cat-sacB (GSO613) using primers MK0202 and
MK0203 containing regions of homology to the mcaS locus. The PCR product was recombineered into strain NM500 containing a
chromosomal copy of the mini λ red cassette to generate NM500 mcaS::cat-sacB. P1 lysate was generated from the mcaS::cat-sacB
strain and used to transduce MG1655 resulting in MG1655 mcaS::cat-sacB (GSO652). The mini λ red cassette was then moved into
this strain background by P1 transduction creating strain MG1655 mcaS::cat-sacB, mini λ::tet (GSO653). PCR of the McaS-3 and
McaS-8 alleles was generated by overlapping PCR (Ho et al. 1989) using the oligonucleotides listed in Table S2. The PCR products
were recombineered into MG1655 mcaS::cat-sacB, mini λ::tet (GSO653) followed by counter selection on M63-minimal glycerol
sucrose plates to select for the loss of the cat-sacB cassette at 30˚C. Colonies were screened by PCR and sequencing to ensure
incorporation of the corresponding McaS mutations.
Plasmid construction
(i) For construction of plasmid pNDM-McaS-3 the mcaS gene was amplified from MG1655 chromosomal DNA using the primers
pA1/O4_mcaS3 and Bam_mcaS_R. The PCR product was digested with AatII and BamHI and inserted into pNDM-220. This low-
copy –number plasmid expresses McaS-3 under the control of the lac promoter (i.e. IPTG inducible).
31
(ii) For construction of plasmid pNDM-McaS-8 the mcaS gene was amplified from MG1655 chromosomal DNA using the primers
pA1/O4_mcaS8 and Bam_mcaS_R. The PCR product was digested with AatII and BamHI and inserted into pNDM-220. This low-
copy –number plasmid expresses McaS-8 under the control of the lac promoter (i.e. IPTG inducible).
(iii) For construction of plasmid pBAD-csrA3xFLAG the csrA::3xFLAG gene was amplified using genomic DNA of strain
MG1655csrA::3xFLAG as a template and oligonucleotides Pst_SD_csrA_F and Hind_FLAG_R as primers. The PCR product was
digested with PstI and HindIII and inserted into PstI and HindIII restricted pBAD33. This plasmid expresses CsrA::3xFLAG under
control of the araP promoter (i.e. induced by arabinose).
(iv) For Plac-controlled expression (pBR-McaS), McaS was PCR amplified, digested with AatII and HindIII and cloned into the
corresponding sites of pBRplac, a high copy number pBR322-derived vector (Guillier and Gottesman 2006). Mutant derivatives of
pBR-McaS were generated by overlapping PCR as described previously (Ho et al. 1989) using the oligonucleotides listed in Table S2
and cloned into the AatII and HindIII sites of pBRplac. All cloning was performed in E. coli TOP10 cells (Invitrogen). All plasmid
inserts were confirmed by sequencing.
32
Supplemental References
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Gotfredsen M, Gerdes K. 1998. The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family. Mol Microbiol 29:
1065-1076.
Guillier M, Gottesman S. 2006. Remodelling of the Escherichia coli outer membrane by two small regulatory RNAs. Mol Microbiol
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Guzman LM, Belin D, Carson MJ, Beckwith J. 1995. Tight regulation, modulation, and high-level expression by vectors containing
the arabinose PBAD promoter. J Bacteriol 177: 4121-4130.
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