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Supporting information
Formation
of
Streptococcus
pneumoniae
choline-binding
proteinDNA
complexes in vitro. Implications for biofilm development
Mirian Domenech, Susana Ruiz, Miriam Moscoso and Ernesto García
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Experimental procedures
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Strains, growth conditions, and DNA
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The present work involved the use of the previously described non-encapsulated S.
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pneumoniae R6 strain (Hoskins et al., 2001). This strain was grown in CpH8 medium
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supplemented (C+Y medium), or not, with 0.08% yeast extract (Lacks and Hotchkiss,
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1960). The cation content of the CpH8 medium was determined by inductively coupled
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plasma optical emission spectrometry (ICP-OES) analysis. Samples were analyzed
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using an Optima 4300™ DV ICP-OES spectrometer (PerkinElmer Inc., USA) (Instituto
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de Ciencias Agrarias, CSIC, Madrid, Spain). All solutions were prepared with ultrapure
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water (resistivity 18.2 MΩ cm at 25°C) produced by a Milli-Q RG water purification
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system (Millipore, USA).
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The optimal conditions for biofilm formation by pneumococcal cells have been
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previously described (Moscoso et al., 2006). To check for biofilm formation, 50 l of a
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1% solution of crystal violet were added to each plate well. The plates were then
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incubated at room temperature for approximately 15 min, rinsed three times with 200 l
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distilled water, and air dried. Biofilm (crystal violet-stained) formation was quantified
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by solubilizing with 95% ethanol (200 l/well) and then determining the absorbance at
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595 nm (A595). The results show the mean ± standard error of at least four independent
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experiments, each performed in triplicate. For biofilm inhibition assays, all required
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reagents were incubated with the cells from the very beginning of growth, unless stated
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otherwise. The appropriate buffer for each case was then added. Plates were incubated
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at 34C under the conditions described in each experiment.
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Plasmid pGL30 (12 kb), constructed in our laboratory, is a pBR322 derivative
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containing a 7.5 kb BclI-fragment of pneumococcal DNA (García et al., 1985), and has
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been used in previous work on proteinDNA binding experiments (Domenech et al.,
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2013). The chromosomal DNA from Mycobacterium smegmatis mc2155 used in the
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different experiments was a generous gift of B. Galán (Centro de Investigaciones
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Biológicas, CSIC, Madrid). S. pneumoniae DNA was purified as previously described
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(Domenech et al., 2013). Agarose gel (0.7%) electrophoresis was performed as
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described elsewhere (López et al., 1984).
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Purification of CBPs
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CBPs overproduced by E. coli strains, were purified by affinity chromatography on
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DEAE cellulose (Sánchez-Puelles et al., 1992). LytA (García et al., 1987), LytB and
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GFP-LytB (De las Rivas et al., 2002), LytC and LytCE365Q (García et al., 1999; Pérez-
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Dorado et al., 2010), Pce (de las Rivas et al., 2001; Hermoso et al., 2005), and CbpF
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(Molina et al., 2009) were overproduced and purified as previously reported. The
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purification of the cell wall-binding domain of LytA (C-LytA) has been described
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elsewhere (Sánchez-Puelles et al., 1990). A purified preparation of an enzymatically
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inactive LytA (LytAH133A) was kindly provided by Pedro García and Blas Blázquez. The
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enzymatically inactive form of LytB (LytBE564A) containing a Glu564→Ala mutation
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was kindly supplied by Palma Rico and Margarita Menéndez (Departamento de
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Química-Física Biológica, Instituto Química-Física “Rocasolano”, CSIC, Madrid,
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Spain). The 564 position corresponds to that in the unprocessed protein. Pure PspC was
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generously provided by Abiodun D. Ogunniyi (Research Centre for Infectious Diseases,
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School of Molecular and Biomedical Science,University of Adelaide, Adelaide,
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Australia).
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Visualization of proteinDNA complexes
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ProteinDNA complexes were examined using epifluorescence and confocal laser
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scanning microscopy (CLSM). Complexes of GFPLytB and pGL30 were incubated for
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15 min at 4°C in the dark with the trimethine cyanine homodimer dye BOBO™-3
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iodide (570/602) (Invitrogen, Karlsruhe, Germany) — a DNA intercalating fluorophore
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providing enhanced fluorescence upon binding to double-stranded DNA. Observations
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were made either at a magnification of 100× using a Leica DFC360-FX epifluorescence
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microscope, or at 63× using a Leica TCS-SP2-AOBS-UV CLSM equipped with an
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argon ion laser. Images were analyzed using LEICA AF 6000-DFC and LCS software
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respectively.
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Unfixed proteinDNA complexes were placed on Formvar-carbon coated copper
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grids, air dried, and examined using a JEOL JEM 1010 transmission electron
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microscope (Centro Nacional de Microscopía Electrónica, Universidad Complutense,
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Madrid, Spain). For the observation of proteinDNA complexes (supported on mica),
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samples were kindly prepared by María Teresa Rejas (Centro de Biología Molecular,
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CSIC, Madrid, Spain), following an established procedure (Sogo et al., 1976).
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Localization of LytBeDNA complexes in S. pneumoniae biofilms
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To localize LytB-eDNA complexes in S. pneumoniae biofilms using CLSM, the R6
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strain was grown in glass-bottomed dishes (WillCo-dish®, WillCo Wells B.V.,
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Amsterdam, The Netherlands) for up to 5 h at 34°C, as previously described (Moscoso
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et al., 2006). The biofilm was incubated for 1 h at 4°C with anti-LytB serum (raised in
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mice and kindly provided by J. Yuste and B. Corsini) (diluted 1/10 in PBS) followed by
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an incubation of 30 min at 4°C in the dark with Alexa fluor 568-labeled goat anti-mouse
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IgG (diluted 1/500) and 10 M SYTO 9 (Life Technology) in 10 mM Tris-HCl buffer,
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pH8.8. The biofilms were then stained with DDAO [7-hydroxy-9H-(1,3-dichloro-9,9-
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dimethylacridin-2-one)] for 10–20 min at room temperature in the dark. Observations
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were made at a magnification of 63× and zoom 2 as detailed above. Projections were
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obtained in the planes x–y (individual scans at 1 mm intervals) and xz (images at 5 m
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intervals).
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Protein techniques
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The purity of CBPs was determined by SDS-polyacrylamide gel electrophoresis
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(Laemmli, 1970) in 10% polyacrylamide gels, and protein bands were visualized by
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staining with Coomassie brilliant blue R250. To test for the absence of nucleases, the
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diverse CBP preparations (5 g) were incubated with pGL30 (100 ng) in PBS
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containing 10 mM MgCl2 for 1 h at 37°C. Then, proteinase K (100 g/ml) was added
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and incubation proceeded for one additional hour. Portions of the mixtures were
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analyzed by 0.7% agarose gel electrophoresis (see above).
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The predicted isoelectric points for proteins and peptides were calculated using either
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of two programs available at (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::iep or
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http://isoelectric.ovh.org/). The pLytB-derived peptides were synthesized using a Focus
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XC automated peptide synthesizer (AAPPTec, Lousville, KY, USA) applying standard
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solid phase Fmos protocols. The following 25/26-amino acid-long synthetic peptides,
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derived from the 635-amino acid-long mature LytB were used in DNApeptide binding
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experiments: P_LytB1 (578-KGILGATKWIKENYIDRGRTFLGNK-602; predicted pI
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10.8 ); P_LytB2 (569-ENYIDRGRTFLGNKASGMNVEYASD-613; predicted pI 4.5);
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P_LytB4 (528-HINALYLLAHSALESNWGRSKIAKDK-553; predicted pI 10.1).
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Numbers correspond to amino acid positions in mature LytB. Amino acid residues
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overlapping in peptides P_LytB1 and P_LytB2 are shown underlined. Peptide P_LytB3
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(DIGGNKTLLKKWRIFITRNYAGGKE; predicted pI 10.8) has the same amino acid
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composition as P_LytB1, but its primary structure was randomly generated
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(http://web.expasy.org/randseq/). Peptide P_LytB5
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(HINALYLLAHSALASNWGRSKIAKDK; predicted pI 10.6) is identical to P_LytB4
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except that the essential catalytic residue Glu-541 (Bai et al., 2014), which is
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highlighted in a black background and corresponds to Glu-564 in the unprocessed
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protein, was substituted by Ala.
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Statistical analysis
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The data for biofilm formation include the mean ± standard error of at least three
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independent experiments, each performed in triplicate. Statistical significance was
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examined using the Student t test. Differences were considered statistically significant
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when P <0.05.
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