Supporting Information

Supporting Information Jennings et al. 10.1073/pnas.1503058112 SI Materials and Methods Bacterial Strains and Culturing. The bacterial strains used in this study

are listed in Table S1. LPS sugar nucleotide allelic replacement strains were constructed using an unmarked, nonpolar deletion strategy described previously (39). Planktonic cultures were routinely grown on Jensen’s chemically defined medium at 37 °C with constant shaking (225 rpm) unless indicated otherwise. Jensen’s medium contained NaCl (85.6 mM), K2HPO4 (14.4 mM), sodium glutamate (92 mM), valine (24 mM), phenylalanine (8 mM), glucose (70 mM), MgSO4 (1.33 mM), CaCl2 (0.14 mM), FeSO4 (0.0039 mM), ZnSO4 (0.0085 mM). The first five ingredients were mixed, the pH was adjusted to 7.3, and resulting solution was subsequently autoclaved. Jensen’s medium was supplemented with glucose and metals after autoclaving. Glucose minimal media (pH 7) was made by modifying the Jensen’s glucose recipe as follows: the glucose concentration was reduced to 0.3 mM, ammonium sulfate was added (15.1 mM final concentration), and the amino acids valine, phenylalanine, and glutamic acid were removed. Media for induced strains was supplemented with arabinose at 0.5% (vol/vol). Pel Polysaccharide Purification. Secreted Pel was prepared for glycosyl composition and linkage analysis by inoculating 50 mL of Jensen’s medium with 0.5 mL of overnight culture of PAO1 ΔwspF Δpsl PBADpel (herein designated PBADpel) and PAO1 ΔwspF Δpsl Δpel (herein designated Δpel). Cultures were grown for 20 h at 37 °C with constant shaking. The supernatant was harvested by centrifugation (8,300 × g for 15 min at 22 °C). Polysaccharides were precipitated for 1 h at 4 °C with ethanol (final concentration 75% vol/vol). The precipitate was washed three times with 95–100% (vol/vol) ethanol, resuspended in 2 mL of buffer (1 mM CaCl2 and 2 mM MgCl2 in 50 mM Tris, pH 7.5), and treated with 5 mg DNase I and 5 mg RNase A for 2 h at 37 °C, followed by 5 mg of proteinase K overnight at 37 °C. Lyophilized samples were submitted to the CCRC at the University of Georgia. Crude secreted Pel in culture supernatant was collected from the centrifugation of overnight cultures. Purified secreted Pel was prepared by cation exchange of culture supernatant. Filtered culture supernatant from PBADpel and Δpel was applied to a 1-mL HiTrap SP-FF strong cation exchange column (GE Healthcare) preequilibrated with 50 mM acetic acid buffer at pH 5.5. The column was washed with equilibration buffer, and Pel was eluted with buffer containing 50 mM acetic acid and 1.25 M NaCl at pH 5.5. Cell-associated Pel was prepared by extraction with EDTA. PBADpel and Δpel cells were harvested by centrifugation from overnight cultures grown at 37 °C with constant shaking. Cell pellets were resuspended in EDTA (0.5 M, pH 8) and heated to 95 °C for 20 min to remove the polysaccharide from the cell surface. Extracts were centrifuged, and the resulting supernatant was retained and thereafter referred to as cell-associated Pel. Immunoblot Analysis. Cell lysates for adsorption of Pel antiserum were generated from strains PA14 ΔpelF and PAO1 ΔwspF Δpsl ΔpelF grown to late log phase in 100 mL of LB medium. Cells were harvested by centrifugation (5,000 × g for 15 min at 4 °C). Pelleted cells were resuspended in 3 mL of buffer (50 mM Tris, 10 mM EDTA, pH 8). Cells were lysed by three freeze/thaw cycles, sonicated at full power for four periods of 20 s on ice, and finally centrifuged (12,000 × g for 15 min at 4 °C). In addition, a third lysate was produced by an ethanol precipitation of the PA14 ΔpelF supernatant, followed by DNase I, RNase A, and proteinase K treatment to simulate conditions under which the Pel antiserum was created. The Pel antiserum was adsorbed for 3–4 h at room temJennings et al. www.pnas.org/cgi/content/short/1503058112

perature with constant rotation using the following reaction mixture: 25 μL of α-Pel antiserum, 33 μL of PA14 ΔpelF lysate, 33 μL of PAO1 ΔwspF Δpsl ΔpelF lysate, and 33 μL of ethanol- precipitated PA14 ΔpelF lysate in 300 μL of 5% (wt/vol) nonfat milk in TBST buffer (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20). Pel immunoblots were conducted essentially as described by Colvin et al. (30). In brief, samples were treated with proteinase K (60 °C for 60 min, followed by 80 °C for 30 min) before blotting 5 μL on a nitrocellulose membrane. Membranes were blocked with 5% (wt/vol) dry skim milk in TBST for 1 h at room temperature. The membrane was probed with α-Pel at a 1:1,000 dilution for 1 h at room temperature. Blots were washed twice for 5 min and once for 10 min with TBST, probed with goat α-rabbit HRP-conjugated secondary antibody (Thermo Scientific) for 1 h at room temperature, and then washed again. The signal for all immunoblots was developed using a SuperSignal West Pico chemiluminescent detection kit (Thermo Scientific) following the manufacturer’s recommendations. The α-PNAG immunoblotting was conducted as follows. Cellassociated Pel (5 μL) from EDTA-treated cell pellets of PBADpel and Δpel cultures was blotted on nitrocellulose membranes and dried for 5 min. Membranes were blocked for 1 h with 5% (wt/vol) skim milk in PBST (PBS containing 0.05% Tween 20). The blots were probed for 1 h with murine IgM MAb 2F3.1D4 (α-PNAG, diluted 1:10,000 in PBST plus 1% BSA) raised against Escherichia coli PNAG. Membranes were washed twice for 5 min and once for 10 min with PBST and treated with the secondary antibody (HRPconjugated anti-murine Ig) at a 1:20,000 dilution for 1 h and then washed again before developing. The α-chitosan immunoblots were created by blotting a cationexchange purification of culture supernatant (5 μL) from PBADpel and Δpel cultures onto a nitrocellulose membrane and then blocking with 3% (wt/vol) skim milk in PBST buffer for 1 h. Blocked membranes were probed with IgM, mAbG7 (α-chitosan, 1:1,000 dilution of 1 μg/mL stock) in PBST containing 1% BSA. After washing, the membrane was treated for 1 h with the secondary antibody used for α-PNAG at a 1:10,000 dilution in PBST. Size Exclusion Chromatography. An overnight culture of PBADpel and Δpel was grown on Jensen’s media at 37 °C with constant shaking. The supernatant was separated from the cell pellet by centrifugation. Cell-associated Pel was purified from the cell pellet by EDTA extraction. Filtered EDTA extract and filtered supernatant were applied to a HiPrep 16/60 Sephacryl S-200 HR size-exclusion column (GE Healthcare) preequilibrated with water to obtain a rough estimate of size. The average molecular weight of secreted Pel was determined by separation of PBADpel supernatant and dextran (Sigma-Aldrich) and cellobiose (Fluka) standards on a Superdex 75 10/300 GL size-exclusion column (GE Healthcare) preequilibrated with water and 0.02% sodium azide at room temperature. The void volume of the column was estimated using filtered blue dextran (2,000 kDa). Fractions (2 mL) were assayed using (i) Pel antiserum to detect Pel and (ii) a phenol-sulfuric acid assay to detect total carbohydrate in the standards. The size of secreted Pel was interpolated from a plot of the partition coefficient (KAV) versus the molecular weight (log scale) of standards. KAV is defined as (Ve − Vo)/(Vt − Vo), where Ve is the elution volume, Vo is the column void volume, and Vt is the total bed volume. Phenol-Sulfuric Acid Assay for Total Carbohydrate. Neutral sugars were quantified using a phenol-sulfuric acid colorimetric assay adapted for a microtiter plate. For this, 25 μL of sample or standard was added to a clear, untreated, flat-bottomed 96-well plate (Nunc) 1 of 9

on ice. In the fume hood, 25 μL of 5% (wt/vol) phenol was added to each well and mixed at slow speed on a vortex mixer for 30 s. Then the plate was returned to the bed of ice, and 125 μL of concentrated sulfuric acid was added, followed by mixing at slow speed for 30 s. The reaction was incubated at 80 °C for 30 min and at room temperature for 5 min. Hexoses were detected by measuring absorbance at 490 nm. Ion-Exchange Chromatography. For a preliminary assessment of the charge of Pel, filtered culture supernatant from PBADpel and Δpel was applied to strong anion- and cation-exchange spin columns (Pierce) equilibrated with 25 mM Tris·HCl pH 7.7 and 25 mM acetic acid pH 5.5, respectively, at room temperature. Pel was eluted with buffer containing 5 M NaCl. Spin fractions were evaluated on α-Pel immunoblots. The minimum salt concentration required to elute Pel (1.25 M NaCl) was determined using a Atka FPLC system (GE Healthcare) to apply a salt gradient (0–5 M NaCl) to a 1-mL HiTrap SP FF strong cation-exchange column (GE Healthcare) equilibrated with 50 mM acetic acid buffer at pH 5.5 at room temperature and loaded with filtered supernatant. To estimate the isoelectric point of Pel, a pH gradient was applied from 5.5 to 8.1 until Pel eluted. Fractions were evaluated with Pel antiserum. Glycosyl Sugar Composition and Linkage Analyses. Glycosyl composition analysis was performed at the CCRC by combining GC/MS of the per-O-trimethyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. More specifically, samples (300–500 μg) were dialyzed and subjected to two different glycosidic cleavage conditions: (i) typical glycosidic cleavage, which did not include any acid hydrolysis before methanolysis, and (ii) strong glycosidic cleavage, which involved hydrolysis with 2 M TFA for 6 h and 6 M HCl for 4 h. Methyl glycosides were prepared from the dry samples by methanolysis in 1 M HCl in methanol at 80 °C for 17 h, followed by re–N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples were perO-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80 °C for 0.5 h. GC/MS analysis of TMS methyl glycosides was performed on an Agilent 6890N GC interfaced to a 5975B MSD, using an Agilent DB-1 fused silica capillary column (30 m × 0.25 mm i.d.) and inositol as an internal standard. Glycosyl linkage analysis was conducted at the CCRC. Samples (1 mg) were acetylated with pyridine/acetic anhydride, dried, suspended in DMSO (200 μL), and stirred for 2 d. Samples were permethylated by treatment with sodium hydroxide for 15 min, followed by methyl iodide in dry DMSO for 45 min. The hydroxide and methyl iodide treatment was repeated to ensure complete methylation of the polymer. The permethylated material was hydrolyzed using 2 M TFA (2 h in a sealed tube at 121 °C), reduced with NaBD4, and acetylated using acetic anhydride/ pyridine. The resulting partially methylated alditol acetates were analyzed by the same GC/MS used for composition analysis. Separation of neutral sugars was performed on a 30-m Supelco 2331 bonded-phase fused silica capillary column. Amino sugars were separated on a Zebron ZB1-MS column. Purification of PelF. The nucleotide sequence of pelF from

P. aeruginosa PA01 was used to design primers specific for the gene that encodes for the full-length protein. The forward primer contains an NdeI restriction site, whereas the reverse primer contains an XhoI restriction site. The amplified PCR products were digested with NdeI and XhoI restriction endonucleases and then cloned into a pET28a vector (Novagen). The fidelity of the nucleotide sequence was verified using DNA sequencing (ACGT DNA Technologies, Toronto, Canada). The resulting expression vector encodes residues 1–507 of PelF fused to a cleavable N-terminal His6 tag for purification purposes. Jennings et al. www.pnas.org/cgi/content/short/1503058112

Expression of PelF was achieved through the transformation of the vector into E. coli BL21 (DE3)-competent cells, which were grown in 4 L of LB medium containing 50 μg/mL kanamycin at 37 °C. The cells were grown to an OD600 of 0.6, after which protein expression was induced by the addition of isopropyl-β-D-1-thiogalacto pyranoside to a final concentration of 1.0 mM. The induced cells were incubated for 20 h at 25 °C before being harvested via centrifugation (6,260 × g for 20 min at 4 °C). The resulting cell pellet was stored at −20 °C until needed. To purify PelF, the cell pellet from 4 L of bacterial culture was thawed and resuspended in 160 mL of buffer A [50 mM Tris·HCl pH 7.5, 300 mM NaCl, 10% (vol/vol) glycerol, 1 mM DTT] containing one SigmaFAST protease inhibitor EDTA-free mixture tablet (SigmaAldrich). Because of the presence of six cysteines in PelF, DTT was included to prevent intermolecular cross-linking of the protein. These cysteines are not expected to be involved in disulfide bond formation, owing to the cytoplasmic localization of the native protein. The resuspension was then lysed by homogenization using an EmulsiFlexC3 homogenizer (Avestin) at a pressure of 10,000–15,000 psi, until the resuspension appeared translucent. Insoluble cell lysate was removed by centrifugation (25,000 × g for 45 min at 4 °C). The supernatant was loaded onto a 5-mL Ni2+-NTA column preequilibrated with buffer A containing 5 mM imidazole to reduce background binding. To remove any contaminants, the column was washed with 10 column volumes of buffer A containing 20 mM imidazole. Bound protein was eluted from the column with five column volumes of buffer A containing 250 mM imidazole. Fractions containing PelF were pooled and concentrated to a volume of 2 mL by centrifugation (2,200 × g at 4 °C) using an Amicon Ultra centrifugal filter device (EMD Millipore) with a 30-kDa molecular weight cutoff. PelF was further purified and buffer- exchanged into buffer B [20 mM Hepes pH 7.5, 150 mM NaCl, 10% (vol/vol) glycerol] with 1 mM DTT by size-exclusion chromatography using a HiLoad 16/60 Superdex 200 gel-filtration column (GE Healthcare). PelF eluted as a single Gaussian peak; all PelF-containing fractions were pooled, and the protein was concentrated by centrifugation (2,200 × g at 4 °C) using an Amicon Ultra centrifugal filter device (EMD Millipore) with a 30kDa molecular weight cutoff and stored at 4 °C. SDS/PAGE analysis revealed that the resulting PelF was ∼95% pure and appeared at its expected molecular weight of 56 kDa. Isothermal Titration Calorimetry. PelF protein samples and nucleosides or nucleotide sugars were prepared in buffer B with 10 mM DTT, and each solution was degassed before experimentation. Isothermal titration calorimetry (ITC) measurements were performed with a microcalorimeter (VP-ITC; MicroCal). Titrations were carried out with 3 mM ligand in the syringe and 60 μM PelF in the cuvette. Each titration experiment consisted of 25 10-μL injections with an 180-s interval between each injection at 10 °C. The ITC data were analyzed using Origin version 5.0 (MicroCal) and fitted using a single-site binding model. Enzyme Digestion of Pellicles. PBADpel pellicles were generated for enzyme digestion by inoculation of 3 mL of Jensen’s medium with 3 μL of overnight culture, followed by static incubation at 37 °C for 4 d. Pellicles were washed to remove spent media and resuspended in buffer with and without enzyme. Enzyme digestion buffer contained 50 mM sodium acetate at pH 5 for cellulose and pH 5.5 for chitosanase. Sodium azide (0.02%) was included in the buffer to reduce bacterial growth. Enzyme concentrations used were 2 mg/3 mL for cellulase (Sigma-Aldrich; C1184, from Aspergillus niger) and 1 μL/3 mL for chitosanase (EMD Millipore; 220477, from Streptomyces sp. N174). Pellicle integrity was evaluated by visual inspection and α-Pel immunoblot of the enzyme/buffer mixture over time. Flow Cell Biofilms and Confocal Microscopy. Biofilms were cultivated in flow cell chambers essentially as described by Colvin et al. (16) 2 of 9

with some modifications. Flow cells were inoculated from a mid-log LB culture that was diluted with glucose minimal media to an OD600 of 0.05 for PA14 and 0.01 for all other strains. Strains that aggregated in mid-log phase were homogenized before dilution for inoculation. Cells were allowed to attach under static conditions to an inverted flow cell for 1 h before induction of flow. Biofilms were grown on glucose minimal media for 1–7 d at room temperature under a constant flow rate (10 mL/h). Biofilms were stained for 15 min with fluorescein-labeled WFL lectin (100 μg/mL; Vector Laboratories) for Pel, TRITC-labeled HHA (100 μg/mL; EY Laboratories) for Psl (25), Syto62 red fluorescent nucleic-acid stain (5 μM; Life Technologies) for biomass, and PI (30 μM; Life Technologies) for eDNA. After staining, flow cells were washed with media at 10 mL/h for 5 min and then visualized on a Zeiss LSM 510 scanning confocal laser microscope. Image analysis was conducted using Velocity software (Improvision). Microscopy images were artificially colored as follows: Pel WFL lectin, red; Psl HHA lectin, blue; Syto62 biomass, green; and PI eDNA, yellow. To determine whether exogenous DNA bound Pel in biofilms, salmon sperm DNA (5 mg/mL; USB) was added to 2-d PBADpel and PBADpsl biofilms. The mature biofilms were incubated statically with the DNA for 15 min, washed with 1.5 mL of glucose minimal media at pH 6.3, stained with PI and Syto62, washed again, and then imaged on the confocal microscope. To ensure that the microscopy images shown in Fig. 3 were representative, line profiles were generated as follows. A horizontal cross-sectional image was captured in the biofilm stalk (bottom quarter of the microcolony), and a 5-μm-wide line was drawn through the microcolony in the cross-sectional image. The normalized fluorescence signal intensity (100 × [intensity/maximum intensity]) was averaged from 12–16 microcolonies from at

least two independent experiments and then plotted against the normalized distance (distance/total length of the microcolony). Gel Electrophoresis. PA14 and PA14 ΔpelC were grown statically in 75 mL of T medium (10 g/L bacto peptone, 5 g/L NaCl) at 30 °C for 6 d. Standing cultures were inoculated with plate-grown bacteria to an OD600 of 0.0025, as recommended by Friedman and Kolter (11). PA14, but not the PA14 ΔpelC, formed pellicle biofilms. Cells were collected by centrifugation, gently washed with water, and extracted with 50% (vol/vol) aqueous phenol at 70–75 °C for 30 min with intensive stirring. The mixture was cooled on ice, and the phases separated by centrifugation (9,000 × g for 15 min). The water phase was further deproteinated by an additional extraction with 90% (vol/vol) phenol, followed by two extractions with chloroform. The phenol extracts were analyzed by sodium deoxycholate (DOC)-PAGE, as described previously (29), and by electrophoresis on 2% (wt/vol) agarose gels (75 mA constant voltage). DOC-PAGE gels were stained with alcian blue (0.005% in fixing solution of acetic acid-ethanol-water 5:40:55) overnight, and with ethidium bromide. In Vitro Pel/Anionic Polymer Cross-Linking Experiments. To determine whether Pel bound DNA or the anionic polymers dextran sulfate, hyaluronan, and mucin in vitro, 1.5 mL of supernatant from 24-h PBADpel and Δpel planktonic cultures at a pH of 8.2 was added to anionic polymer (5 mg/mL) and shaken until completely dissolved. Subsequently, the pH of the solution was adjusted to 7.3 or 6.3 with HCl. Congo red was added at a final concentration of 40 μg/mL. Aggregates resulting from the cross-linking of the Pel to anionic polymers were detected by visual inspection.

Fig. S1. α-Pel immunoblot indicates that LPS biosynthesis enzymes and UDP-Glc are not required for Pel production. Cell-associated and secreted (supernatant) Pel extracts from PBADpel (+) and Δpel (−) in a PAO1 ΔwspF Δpsl ΔR2 background were blotted. LPS is a major constituent of the outer membrane in Gram-negative bacteria and is composed of three parts: lipid A, a core containing KDO, and O-antigen polysaccharides (A and B bands). Mutant phenotypes were as follows: ΔwbpX, deficient in A band LPS; ΔwbpJ and ΔwbpM, deficient in B band LPS; ΔwbpL, deficient in A and B bands; ΔalgC, deficient in A band with a truncated LPS core; and ΔgalU, unable to synthesize UDP-Glc, deficient in A and B bands with a truncated LPS core.

Jennings et al. www.pnas.org/cgi/content/short/1503058112

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Fig. S2. PelF, the predicted glycosyltransferase, binds UDP with micromolar affinity. Isothermal titration calorimetry of PelF with the indicated nucleosides. For each nucleoside, the heats of injection (Upper) and normalized integration as a function of the molar syringe and cell concentrations (Lower) are shown. The calculated dissociation constant (KD) is indicated where binding was observed.


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Fig. S3. Size-exclusion calibration curve for molecular weight determination of secreted Pel. The partition coefficient (KAV) versus molecular weight (log scale) of standards and PBAD pel supernatant is shown. KAV is defined as (Ve− Vo)/(Vt − Vo), where Ve is the elution volume, Vo is the column void volume, and Vt is the total bed volume. Error bars represent SD (n = 2).

Fig. S4. Secreted Pel has an overall positive charge. Supernatant from PBADpel, but not from Δpel, bound a strong-cation exchange column at pH 5.5 and eluted at 1.25 M NaCl. Fractions from the cation column were probed with Pel antiserum (Top). Load indicates immunoblot of samples loaded to the column. Results shown are from a step gradient (Bottom), but a continuous gradient of salt concentration was initially used to determine the minimum salt concentration needed to elute Pel.


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Fig. S5. Antibody binding, enzyme digestion, and lectin staining provide supportive evidence that Pel contains partially acetylated GalNAc and GlcNAc. (A) Monoclonal antibodies for chitosan bind secreted Pel from a cation-exchange purification of PBADpel, but not Δpel, culture supernatant. (B) Monoclonal antibodies for PNAG bind cell-associated Pel from EDTA extraction of cell pellets in PBADpel, but not in Δpel. (C) Chitosanase has activity on pellicles. PBADpel pellicles were grown statically before liquid was removed and replaced with buffer (−) or buffer plus enzyme (+). (Upper) Images of enzyme digest taken after 2 d. (Lower) α-Pel immunoblot of enzyme digests show chitosanase results in slow degradation of Pel-antisera signal compared with more rapid degradation by cellulase. (D) WFL, which recognizes GalNAc sugars, specifically binds PBADpel cell clusters (red), but not Δpel cells. A merge of bright field and red channel (Upper) and red channel only (Lower) are shown. (Scale bar: 10 μm.)


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Fig. S6. Localization patterns of Pel are consistent in biofilm time course. (A) Pel lectin was specific for Pel and showed minimal staining in PAO1 ΔwspF Δpel cultivated for 4 d on glucose minimal media. (B) Pel was localized to the stalk with minimal periphery staining in mature PAO1 ΔwspF microcolonies. (C) In PA14, Pel was localized to the stalk and periphery of the mushroom cap. For both strains, Pel staining was uniform in younger microcolonies (days 1–2). Shown are representative confocal images of flow cell biofilms cultivated for 1–4 d before staining with Pel-specific lectin (WFL, red) and biomass stain (Syto62, green). (D) Pel was localized in induced strain to the periphery and stalk, but Psl was uniformly distributed throughout the entire biofilm population. Shown are representative confocal images of flow cell biofilms from PBADpel and PBADpsl cultivated for 2 d in the presence of arabinose before staining with biomass stain (Syto62, green) and Pel-specific lectin (WFL, red) or Psl-specific lectin (HHA, blue). (Scale bars: 30 μm.)


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Fig. S7. (A) Pel and eDNA interfere with staining of each other when stained with WFL and PI simultaneously in the same microcolony (top row) or sequentially with WFL followed by PI (bottom row). Shown are representative confocal images of flow cell biofilms from PAO1 ΔwspF cultivated for 4 d before staining. (Scale bars: 30 μm.) (B) An HMW band is visible in DOC-PAGE gel profiles of the phenol extracts from PA14 cells, but not from ΔpelC cells. The gel was stained with alcian blue. (C) An HMW band in DOC-PAGE gel profile of the phenol extracts from PA14 before (−) and after (+) DNase treatment. The gel was stained with ethidium bromide. A DNA ladder is shown in leftmost lane. (D) Abundant DNA is present in 2% agarose gel profiles of the phenol extracts from PA14 cells, but not from ΔpelC cells.

Table S1. Bacterial strains used in this study Strains

Description

Source

PAO1 PAO1 PAO1 PAO1 PAO1

Wild type wspF, nonpolar mutation wspF, nonpolar mutation; pslBCD, polar mutant of the psl operon arabinose-inducible psl operon wspF, nonpolar mutation; pslBCD, polar mutant of the psl operon; arabinose-inducible pel operon wspF, nonpolar mutation; pelA, polar mutant of the pel operon; pslBCD, polar mutant of the psl operon

(30) (30) (30) This study (30)

ΔwspF ΔwspF Δpsl ΔwspF Δpel PBADpsl ΔwspF Δpsl PBADpel

PAO1 ΔwspF Δpsl Δpel PA14 ΔwspF PA14 PBADpel PA14 PA14 ΔpelF PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl PAO1 ΔwspF Δpsl

Chromosomal replacement of the native promoter with araC-PBAD promoter Wild type ΔwbpJ ΔR2 PBADpel ΔwbpX ΔR2 PBADpel ΔwbpL ΔR2 PBADpel ΔwbpM ΔR2 PBADpel ΔalgC ΔR2 PBADpel ΔgalU ΔR2 PBADpel ΔwbpJ ΔR2 Δpel ΔwbpX ΔR2 Δpel ΔwbpL ΔR2 Δpel ΔwbpM ΔR2 Δpel ΔalgC ΔR2 Δpel ΔgalU ΔR2 Δpel

Deficient Deficient Deficient Deficient Deficient Deficient


in in in in in in

B band LPS and R2 pyocin production (40) A band LPS (32, 40) A and B band LPS (32, 40) B band LPS (32, 40) A band LPS and with a truncated LPS core (31, 40) A and B band and UDP-Glc, and with a truncated LPS core (33, 40)

(30) This study (12) (30) This study This study This study This study This study This study This study This study This study This study This study This study This study

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Movie S1. Pel is a major structural component of the biofilm stalk. In this 3D movie of a PBADpel 3-d biofilm stained with biomass stain (Syto62, green) and Pelspecific lectin (WFL, red), the image rotates about the z-axis initially showing both red and green channels, after which the green channel fades to accentuate columns of Pel in the biofilm stalk. Note how intertwined fibers or columns of Pel connect the surface to the top of the microcolony. Movie S1


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