Supporting information
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Detailed experimental procedures
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Bacterial strains and culture conditions
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All experiments were performed using S. frigidimarina NCIMB400 (Reid and Gordon, 1999).
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S. frigidimarina NCIMB400 is a marine organism isolated from the North Sea near Aberdeen, UK
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(Lee et al., 1977) and the genus model bacterium used to study the electron transfer ability, S.
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oneidensis MR-1 was isolated from the freshwater sediment of Oneida Lake, New York, USA (Myers
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and Nealson, 1988). The Pseudomonas aeruginosa PAO1 strain was used as a test control for all the
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phenotypical assays. All microorganisms were cultured in Luria-Bertani broth (Sigma-Aldrich, St.
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Louis, MO, USA) but Shewanella species growth was performed at 20°C with agitation (120 rpm),
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and P. aeruginosa at 30°C.
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Adhesion assay
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For the adhesion assays, post-exponential S. frigidimarina NCIMB400 and S. oneidensis MR-
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1 strains grown in LB medium were inoculated at the OD600 values of 0.1, 0.3, 0.5, or 0.7, in artificial
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seawater (ASW; (Sigma-Aldrich, St Louis, MO, USA) and Phosphate buffered saline (PBS,) solution
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in black microplates (sterile black polystyrene plates; Nunc, Fisher Scientific, Illkirch, France). After
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incubation for 0, 5, 12, 24 or 48 h, the non-adherent bacteria were removed by three successive washes
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in 36 g/L NaCl solution (Camps et al., 2011). The adhered bacteria were stained with Syto 61 Red
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fluorescent nucleic acid stain (Life Technology, Molecular Probes, Carlsbad, CA, USA). After 10 min,
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excess stain was removed by one wash with the NaCl solution and the fluorescence intensity (FI) was
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measured (λ exc= 615 nm, λ em= 670 nm) (Infinit 200 microplate fluorescence reader; Tecan, Lyon,
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France). FI was calculated per well, as: FI= FI average assay – FI average negative control. These
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experiments were performed in triplicate and repeated 3 times.
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Bacterial biofilm formation assessment using the Biofilm Ring Test ®
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All strains were tested to evaluate their adhesion and biofilm formation abilities to polystyrene
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using the BioFilm Ring Test® (Chavant et al., 2007). Biofilm formation was quantitatively assessed
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according to the manufacturer protocol developed (Biofilm Control, Saint-Beauzire, France). The
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toner solution, TON006N, containing inert paramagnetic microbeads was added (10 µL/mL) to post-
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exponential bacterial suspension at OD600nm of 0.01 in LB broth, ASW or PBS solutions. Eight well
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strips from the Biofilm Ring Test microplates were incubated for 0, 5, 12, 24 and 48 h at 20°C (30°C
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for PAO1) and analyzed before and after magnetization of the well. Mixtures containing the toner
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without bacteria were used as negative controls. Because of the important presence of salts, the non
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ionic surfactant Tween 20 was used at 0.004% (m/v) in the ASW and PBS solutions. We have verified
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that no growth was observed at this concentration of Tween in both solutions (data not shown). The
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biofilm capacity of each strain was expressed as ΔBFI corresponding to BFI (Biofilm formation index)
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control – BFI sample. In parallel, strips inoculated with post-exponential S. frigidimarina NCIMB400,
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S. oneidensis MR-1 and P. aeruginosa PAO1 at OD600nm 0.01, were washed at 24 h, and the biofilm
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containing bacteria was scraped and resuspended in sterile LB, ASW and PBS solutions in order to
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evaluate their relative concentrations at OD600nm and after counting CFUs. These experiments were
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performed in triplicate and repeated 3 times.
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Preparation of sessile and planktonic samples of S. frigidimarina NCIMB400 for the
proteomic study
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Post-exponential S. frigidimarina NCIMB400 were grown in LB medium, and the suspension
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diluted to OD600 0.1. Planktonic cells were grown in a 250 mL Erlenmeyer flask containing 100 mL of
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cell suspension incubated at 20°C with agitation (120 rpm). Sessile biomass was produced in 10 Petri
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dishes containing 10 mL of cell suspension incubated at 20°C without agitation. After 24 h of growth,
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planktonic cells were centrifugated (3 000 g, 10 min at 20°C). For the production of sessile bacteria,
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non-adherent cells were washed off, and adhered cells were harvested in 10 mL LB with a sterile
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scraper. Pellets were washed twice by centrifugation and resuspended in 10 mL 40 mM Tris-HCl
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buffer (pH 8). Cell suspensions were immediately stored at -20°C until analysis.
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Protein extraction
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Cell suspensions were thawed, centrifuged and pellets were used to produce two types of non-
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soluble protein fractions, called “whole non-soluble protein faction” and “membrane-enriched non-
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soluble protein fraction” (Table 1). Whole non-soluble protein fraction was extracted by
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homogenizing bacterial cells with an extraction solution consisting of 7 M urea, 2 M thiourea, 1 % w/v
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CHAPS, 1 % w/v DTT, 0.2 % v/v carrier ampholytes, 0.002 % v/v Bromophenol Blue, 3 % v/v Triton
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X-100 on the basis of 4 ml of extraction solution per g of wet biomass. The mixture obtained was
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incubated for 1 h at room temperature (stirring for 30 s every 15 min) and then sonicated on ice, using
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a microtip Vibra Cell 734 24 (Bioblock Scientific, Illkirch, France), during 5 min (10 s on/ 10 s off, 20
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kHz and 50 W) and centrifuged (10 min at 15 000 g, 20°C). The pellet was stored at -80°C until
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analysis. Part of this fraction was subjected to a membrane protein enrichment step. Membrane-
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enriched non-soluble protein fraction was extracted using the ReadyPrep Protein Extraction
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(Membrane I) Kit (Bio-Rad, Hercules, CA, USA). Protein extracts were processed using the
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ReadyPrep 2-D Clean-up Kit (Bio-Rad, Hercules, CA, USA) and solubilized in the same extraction
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solution (see above). Protein determination of the extracts was achieved using the Reagent Compatible
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Detergent Compatible Protein Assay (RC DC Protein Assay, Bio- Rad, Hercules, CA, USA) and
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bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA) as the standard. Fractions were stored at -
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80°C until analysis.
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2D gel electrophoresis
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Isoelectric focusing (IEF) was carried out according to O’Farrell et al. (O'Farrell, 1975), using
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a pre-prepared immobilized pH gradient (IPG) strip (17 cm length, linear gradient, pH 4-7 and pH 3-
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10) from Bio–Rad, Hercules, CA, USA, under which a sample (350 µL) containing 120 µg proteins,
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was transferred. Rehydration and subsequent IEF of the protein extract were performed in the
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horizontal electrophoresis system PROTEAN IEF Cell (Bio–Rad, Hercules, CA, USA), in the
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following manner: 18 h at 50 V (active rehydration), 2 h at 100 V, 2 h at 250 V, 2 h at 500 V, 2 h at
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1 000 V, 2 h at 4 000 V and 5 h at 10 000 V, and 6 h at 500 V, so as to reach a total minimal value of
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60 000 V.h for each loaded IPG strip. Rehydration and all steps of IEF were run at 20°C. After
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focusing, the IPG strips were successively equilibrated for 10 min at room temperature in equilibration
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buffers 1 (6 M urea, 2 % w/v SDS, 0.375 M Tris-HCl pH 8.8, 20 % v/v glycerol, 2 % w/v DTT) and 2
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(6 M urea, 2 % w/v SDS, 0.375 M Tris-HCl pH 8.8, 20 % v/v glycerol, 2.5 % w/v iodoacetamide).
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Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) was performed
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according to Laemmli et al. (Laemmli, 1970). The equilibrated IPG strips were placed in dyed
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(Bromophenol Blue) melted agarose, across a handmade polyacrylamide gel (18 cm × 18 cm × 1 mm,
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stacking gel: 5 %, resolving gel: 12 %). A volume of 20 µL of molecular weight marker solution (10 –
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250 kDa range, Precision Plus Protein Standards Dual Color, Bio-Rad, Hercules, CA, USA) was
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loaded at the left top of the gel. The gels were run at + 4°C in a Protean II XL (Bio-Rad, Hercules,
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CA, USA), with a constant current of 20 mA per IPG strip for 1 h and subsequently with a constant
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current of 30 mA per IPG strip until the dye reached the bottom of the gel. Following SDS-PAGE,
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each 2D-gel was stained by submersion during 1 h 30 min in 150 mL of sensitive Coomassie Brilliant
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Blue (Imperial Protein Stain from ThermoScientific, Waltham, MA, USA), followed by overnight de-
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staining in 150 mL of ultrapure water, until visualization of protein spots. Each 2D gels presented in
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this study was representative of two gels performed with the same sample. At least, three independent
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2D electrophoresis experiments were performed for each condition. The protein spots on the gels were
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analyzed using the PDQuest 2D-Analysis software version 8.0.1. (Bio-Rad, Hercules, CA, USA).
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Manual editing and normalization were performed after automated spot detection. Gel annotations and
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matching fidelity were verified manually.
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Protein identification by liquid chromatography tandem mass spectrometry
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Protein spots selected for LC-MS/MS analyses presented statistically significant variations
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(p<0.05), and an average fold change equal or higher than 2 in absolute value between sessile and
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planktonic conditions (except for spots 19, 20 and 22). Spots were excised from the Coomassie Blue-
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stained gels with a cut tip and each of them was added to an Eppendorf microtube. Protein
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identification was carried out at the “Plateforme d’Analyse Protéomique de Paris Sud-Ouest”
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(PAPPSO) platform (INRA, Jouy-en-Josas, France) using an Ultimate 3000 LC-MS/MS system
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(Dionex, Voisins le Bretonneux, France) (http://pappso.inra.fr).
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In brief, gel plugs were first washed in different solutions of 50 % v/v acetonitrile and 100 %
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v/v acetonitrile supplemented with DTT for 1 h at 56°C. Samples were then incubated in 25 mM of
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iodoacetamide for 1 h at room temperature, in the dark. Digestion was performed for 3 h at 37°C with
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125 ng of modified trypsin (Promega, Fitchburg, WI, USA). Tryptic peptides were first extracted with
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solution of 50 % and 100 % acetonitrile. Both peptide extracts were pooled, dried in a vacuum-speed
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concentrator, and suspended in 25 L of 2% v/v acetonitrile and 0.08 % v/v trifluoroacetic acid in
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water. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed with
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an Ultimate 3000 LC system (Dionex, Voisins-le-Bretonneux, France) connected to an LTQ-Orbitrap
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Discovery mass spectrometer (Thermo Fisher) via a nanoelectrospray ion source. Ionization (1.5 kV
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ionization potential) was performed with liquid junction and a non-coated capillary probe (10 µm
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inner diameter; New Objective). Peptide ions were analysed using Xcalibur 2.07 with the following
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data-dependent acquisition steps: (1) full MS scan (mass-to-charge ratio (m/z) 300 - 1 400, centroid
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mode) and (2) MS/MS (qz = 0.22, activation time = 50 ms, and collision energy = 35%; centroid
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mode). Step 2 was repeated for the three major ions detected in step 1. Dynamic exclusion was set to
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30 s. Results were analyzed using the X!TandemPipeline software version 3.2.
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qRT-PCR
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Planktonic and sessile bacteria were harvested, centrifuged at 6 000 g for 15 min and
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resuspended at 109 cells/mL in the RNA Protect Bacteria Reagent (Qiagen, Hilden, Germany). Pellets
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were stored in -80°C for maximum one month. RNA extraction was performed using NucleoSpin
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RNA II (Macherey-Nagel, Düren, Germany) as described by the manufacter. cDNAs were obtained
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using PrimeScriptTM RT Master (Perfect Real time) Mix (Takara, Otsu, Shiga, Japan) and were
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stored at -20°C. Real time PCR from the cDNA samples was performed using a LightCycler 480
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(Roche life Science, Penzberg in Upper Bavaria, Germany). The 16S rRNA gene was used as a
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housekeeping gene and a no template control (NTC) was used as negative control. Primers for the 16S
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rDNA reference gene were qPCR_NCIMB400_16S_FW (5′TTT-AAC-CTT-GCG-GCC-GTA-CT-3′)
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and qPCR_NCIMB400_16S_REV (5′ACT-GAC-GCT-CAT-GCA-CGA-AA-3′), primers for the hcp1
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gene were qPCR_Hcp1_Fw (5′-CAA-CTC-GTT-TAG-GTG-ACG-TGA-CCA-3′) and
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qPCR_Hcp1_Rev (5′-CGG-TCA-CCA-TGA-ACT-GAA-ACA-CTG-3′), for the impB gene were
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qPCR_ImpB_Fw (5′-ATC-GAT-CGC-GAC-AAC-TTC-AAT-G-3′) and qPCR_ImpB_Rev (5′-ACA-
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ATA-GCT-GCA-GGC-TCG-AAG-T-3′), for the pep gene were qPCR_PEP_Fw (5′- TTC-GCA-CCT-
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GTG-ACG-GAT-GTT-′3) and qPCR_PEP_Rev (5′- AGG-GCC-TAA-ATC-GGG-AAT-TTG-CAC-
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′3), for the flgE gene were qPCR_FlgE_Fw (5′-AGT-GGC-GGT-GGC-TTC-TTT-GTT-A-3′) and
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qPCR_FlgE_Rev (5′-ACC-ATC-TTC-ATC-CAC-TGG-AAA-ACC-3′).
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Statistical analysis
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GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis
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of the adhesion and biofilm formation data (excluding the test control PAO1). Data were analyzed
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using one-way and two-way ANOVA and treatment effects separated using the Bonferroni and
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Turkey’s multiple comparison post-hoc test. Statistical significance was accepted at p < 0.05.
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Supplementary data
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Table S1. Number of protein spots of non-soluble and membrane-enriched fractions, separated
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by 2D-eletrophoresis comparing planktonic and sessile conditions
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Membrane-enriched non-
Membrane-enriched non-
soluble fraction (pH 3-10)
soluble fraction (pH 4-7)
Whole non-soluble fraction
Planktonic
Sessile
Planktonic
Sessile
Planktonic
Sessile
Total
105
267
167
185
564
416
Matched
72
72
137
137
304
304
Up-regulated
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66
79
58
191
113
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7
8
9
10
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7
A
B
21
C
D
19
18
8
1
16
2
9
13
20
22
10
17
7
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Figure S1. 2D polyacrylamide gels of planktonic and sessile membrane protein enriched
fractions from S. frigidimarina NCIMB400, performed using pH 3-10 (A and B) or pH 4-7 IPG
strips (C and D). Up-regulated spots obtained under sessile conditions (B and D) are indicated by
arrows. Spot numbers in D correspond to those displayed in Table 1. The pI and molecular weight
(MW) scales are indicated for the horizontal and vertical axis, respectively.
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