Raffinose, a plant galactoside, inhibits Pseudomonas aeruginosa
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1 Raffinose, a plant galactoside, inhibits Pseudomonas aeruginosa biofilm formation via binding to LecA and decreasing cellular cyclic diguanylate levels Han-Shin Kim 1, Eunji Cha 1, YunHye Kim 2, Young Ho Jeon 2, Betty H. Olson 3, Youngjoo Byun 2* and Hee-Deung Park 1* 1-School of Civil, Environmental and Architectural Engineering, Korea University, Anam-Dong, Seongbuk-Gu, Seoul , South Korea 2-College of Pharmacy, Korea University, Sejong-ro 2511, Jochiwon-eup, Sejong, 339-7, South Korea 3-Department of Civil and Environmental Engineering, University of California, Irvine, CA 92697, USA
2 Figure S1 a 7 Biofilm formation (OD545nm/OD595nm) Raffinose b c Biofilm formation (OD545nm/OD595nm) Sucrose Biofilm formation (OD545nm/OD595nm) Galactose Fig. S1. Effects of raffinose on P. aeruginosa biofilm formation. Biofilm formation at different concentrations (,.1, 1, 1, 1, and 1) of raffinose (a), sucrose (b), and D-(+)-galactose (c) for 24 h in microtiter plates. Error bars indicate the standard deviations of 1 measurements., P <.5 versus the control. *, P <.5 versus the control.
3 Figure S2 a OD49nm/OD595nm Planktonic Biofilm b OD75nm/OD595nm *. Planktonic Biofilm Fig. S2. Effect of raffinose on EPS production. (a) Total carbohydrate in EPS of P. aeruginosa planktonic and biofilm cells cultured with different concentration of raffinose (, 1, 1, 1, and 1, µm) for 24 h. (b) Total protein in EPS of P. aeruginosa planktonic and biofilm cells cultured with different concentration of raffinose (, 1, 1, 1, and 1,) for 24 h. Error bars indicate the standard deviations of 3 measurements., P <.5 versus the control. *, P <.5 versus the control.
4 Figure S3 Swimming motility (.3% AB agar) Twitching motility (1.% AB agar) DMSO 1 DMSO Fig. S3. Swimming and twitching motility of P. aeruginosa cultured with different concentrations of ra ffinose (, 1, 1, 1, and 1,) for 24 h on AB agar plate.
5 Figure S4 raffinose galactose + LecA + raffinose H 1 H 6 galactose + LecA galactose LecA Fig. S4. Monitoring of galactose signals with the treatment of excess raffinose by CPMG NMR spectroscopy. First row: NMR spectrum of raffinose in the absence of LecA. Second row: NMR spectrum of galactose (.1 mm) and LecA (5) with the addition of raffinose (1 mm). Third row: NMR spectrum of galactose (.1 mm) in the presence of LecA (5). Fourth row: NMR spectrum of galactose (1 mm) in the absence of LecA. Fifth row: NMR spectrum of LecA only
6 Figure S5 (a) Raffinose (b) Galactose (c) Sucrose Fig. S5. Isothermal titration calorimetric (ITC) measurements for the binding of the ligands to LecA. Th e raw ITC raw data (first row) and integrated titration curves (second row) of raffinose, galactose, and s ucrose.
7 Figure S6 3 C-di-GMP (pmol/mg protein) Wild type ΔwspF Fig. S6. Effects of raffinose on cellular c-di-gmp levels in P. aeruginosa. Cellular c-di-gmp levels in P. aeruginosa wild type and ΔwspF mutant treated without and with 1 raffinose for 24 h. Error ba rs indicate the standard deviations of 3 measurements., P <.5 versus the control.
8 Figure S7 Released cell (CFU/ml) 5x1 6 4x1 6 3x1 6 2x1 6 1x1 6 Untreated Treated * 2hr 4hr Exposure time Fig. S7. Effects of raffinose on P. aeruginosa biofilm dispersion. Bacterial cells formed biofilm format ion on the glass slide and 1 concentrations of raffinose treats for 2 and 4 h. Released bacteria cel l count assay. Error bars indicate the standard deviations of 3 measurements., P <.5 versus the control. *, P <.5 versus the control.
9 Figure S8 a b 1.8 Wild type ΔlecA Wild type + 1 Raffinose ΔwspF ΔwspF + 1 Raffinose 8 Wild type ΔlecA Relative gene expression levels * leca gene bis-pnpp degradation (OD41mg protein -1 ) Fig. S8. Effects of raffinose on P. aeruginosa leca gene expression and PDE activity. (a) Relative leca gene expression for leca deletion mutant (ΔlecA) and c-di-gmp overproducing mutant (ΔwspF) with a nd without addition of 1 raffinose. (b) PDE activity measurement via bis-pnpp degradation for le ca deletion mutant (ΔlecA). Error bars indicate the standard deviations of 3 measurements., P <. 5 versus the control. *, P <.5 versus the control.
10 Table S1 Table S1. Thermodynamic parameters and K d values of raffinose and galactose from ITC experiments. Ligand ΔH (kcal/mol) -TΔS (kcal/mol) ΔG (kcal/mol) Stoichiometry K d (µm) Raffinose Galactose
11 Table S2 Table S2. RT-qPCR primers used in this study. Primer Target gene Sequence (5 3 ) Tm ( o C) GC % leca-f leca GGG TTG CAC CCA ATA ATG TC leca-r leca CCA ATA TTG ACG CTG AAC GA proc-f proc GGC GTA TTT CTT CCT GCT GA proc-r proc CCT GCT CCA CTA GTG CTT CG Product size (bp) Reference 1 This study 236 Savli H et al. Savli H, et al. (23) Expression stability of six housekeeping genes: A proposal for resistance gene quantificat ion studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol 52(Pt 5):
Supplemental Data. Shin et al. Plant Cell. (2012) /tpc YFP N
MYC YFP N PIF5 YFP C N-TIC TIC Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 Supplemental Figure. TIC interacts with MYC in the nucleus. Bimolecular fluorescence complementation assay using
Culture Density (OD600) 0.1. Culture Density (OD600) Culture Density (OD600) Culture Density (OD600) Culture Density (OD600)
A. B. C. D. E. PA JSRI JSRI 2 PA DSAM DSAM 2 DSAM 3 PA LNAP LNAP 2 LNAP 3 PAO Fcor Fcor 2 Fcor 3 PAO Wtho Wtho 2 Wtho 3 Wtho 4 DTSB Low Iron 2 4 6 8 2 4 6 8 2 22 DTSB Low Iron 2 4 6 8 2 4 6 8 2 22 DTSB
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Supplementary Document 1. Supplementary Table legends 2. Supplementary Figure legends 3. Supplementary Tables 4. Supplementary Figures 5. Supplementary References 1. Supplementary Table legends Suppl.
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Supplementary Materials
Supplementary Materials 1 Supplementary Table 1. List of primers used for quantitative PCR analysis. Gene name Gene symbol Accession IDs Sequence range Product Primer sequences size (bp) β-actin Actb gi
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Citation for published version (APA): Oosterveer, M. H. (2009). Control of metabolic flux by nutrient sensors Groningen: s.n.
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Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.
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