Supplemental Information.docx

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]

1

mailto:[email protected]

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.

5

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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http://mfold.rna.albany.edu/?q=mfold

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.

13

http://mfold.rna.albany.edu/?q=mfold

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


GSO637 MPK0303 PM1205 lacI'::PBAD-pgaA108-lacZ, ∆abgR-ydaL::kan This study




16


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


GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGAGACAATGCCGGATTTAAGTGACGGATGCACTGCTGTG



GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGTGACAATGCCGGATTTAAGACGCGGATGCACTGCTGTG



GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGAGACAATGCCGGATTTAAGTGACGGTTGCACTGCTGTGTGTAC



GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGTGACAATGCCGGATTTAAGTGACGGATGCACTGCTGTG



GAAATTAATACGACTCACTATAGGACCGGCGCAGAGGAGACAATGCCGGATTTAAGACGCGGTTGCACTGCTGTGTGTAC


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

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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


MK0396 pgaA67-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATTCTTTCTTTTCAGTTACCTG


MK0436 pgaA30-lacZ fwd ACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATAGATTTTGGCAATACATGGA


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


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


CGTCTTAAATCCGGCATTGTCACCTCTGCGCCGGT Forward primer for mcaS-8 integration on chromosome; use with MK0160 in PCR #1

28


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

29

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

Cherepanov PP, Wackernagel W. 1995. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed

excision of the antibiotic-resistance determinant. Gene 158: 9-14.

Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc

Natl Acad Sci USA 97: 6640-6645.

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

59: 231-247.

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.

Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain

reaction. Gene 77: 51-59.

Hobbs EC, Astarita JL, Storz G. 2010. Small RNAs and small proteins involved in resistance to cell envelope stress and acid shock in

Escherichia coli: analysis of a bar-coded mutant collection. J Bacteriol 192: 59-67.

Jørgensen MG, Nielsen JS, Boysen A, Franch T, Møller-Jensen J, P. V-H. 2012. Small regulatory RNAs control the multi-cellular

adhesive lifestyle of Escherichia coli. Mol Microbiol 84: 36-50.

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Lange R, Hengge-Aronis R. 1991. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol

Microbiol 5: 49-59.

Mandin P, Gottesman S. 2009. A genetic approach for finding small RNAs regulators of genes of interest identifies RybC as

regulating the DpiA/DpiB two-component system. Mol Microbiol 72: 551-565.

-. 2010. Integrating anaerobic/aerobic sensing and the general stress response through the ArcZ small RNA.. EMBO J 29: 3094-3107.

Overgaard M, Johansen J, Møller-Jensen J, Valentin-Hansen P. 2009. Switching off small RNA regulation with trap-mRNA. Mol

Microbiol 73: 790-800.

Thomason MK, Fontaine F, De Lay N, Storz G. 2012. A small RNA that regulates motility and biofilm formation in response to

changes in nutrient availability in Escherichia coli. Mol Microbiol 84: 17-35.

Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L. 2001. Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci USA

98: 15264-15269.

Zhang A, Schu DJ, Tjaden BC, Storz G, Gottesman S. 2013. Mutations in interaction surfaces differentially impact E. coli Hfq

association with small RNAs and their mRNA targets. J Mol Biol In press.

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