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SUPPLEMENTAL INFORMATION

    Dual function of the McaS small RNA in controlling biofilm formation

    1,42,3,411,52,5Mikkel Girke Jørgensen, Maureen K. Thomason, Johannes Havelund, Poul Valentin-Hansen and Gisela Storz

    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 valentin@bmb.sdu.dk or storzg@mail.nih.gov

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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

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Supplemental Methods Detailed descriptions of strain and plasmid construction. 30

Supplemental References 33

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Supplemental Figure S1. Assays of PM1205 ΔabgR-ydaL derivatives with (A) pgaA-lacZ and (B) pgaA-lacZ fusions transformed 15567

    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.

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    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 OD of ~0.05 in LB and allowed to grow for 1.5 h at 600

    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

    32RNA was extracted and 10 μg was separated on an 8% polyacrylamide-7M urea gel, transferred to a membrane and probed with P-

    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 OD 600

    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.

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    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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