Fig. S1. Analysis of the optimal conditions for LytC–DNA complex formation using agarose gel electrophoresis.

A. Influence of ionic strength. LytC (1 μg) and pGL30 (40 ng) were incubated at 37°C for 1 h in 10 mM Tris-HCl (pH 7.0) with the indicated additions of NaCl or choline chloride (ChoCl).

B. Effect of pH. The experimental conditions were as described in (A) except that the pH of the buffer was adjusted to the values indicated.

C. Effect of incubation temperature. The incubation of LytC plus pGL30 was allowed to proceed for 1 h in 10 mM Tris-HCl buffer (pH 8.5) at the indicated temperatures.

D. pGL30 (40 ng) was incubated with the indicated amounts of LytC for 1 h at 37°C in 10 mM Tris-HCl, pH 7.0.

E. pGL30 (40 ng) and LytC (1 μg) were incubated in 10 mM Tris-HCl buffer (pH 7.0) at 37°C for the indicated times. C, pGL30 control; S, BstEII-digested λ DNA.


Fig. S2. Assay of biofilm degradation by sodium metaperiodate. An R6 biofilm grown for 6 h at 34°C was treated with the indicated amounts of NaIO4 at 37°C for 2 h. Open and filled bars indicate growth and biofilm formation respectively. *P < 0.05; ***P < 0.001.


Fig. S3. Inhibition of Calcofluor binding by cellulose. Biofilms of strain R6 were incubated with a combination of SYTO 59 (A, C and E; red) and Calcofluor (B, D and F; blue). (D) and (F) correspond to Calcofluor staining performed in the presence of 25 mg−1 of cellulose and pullulan respectively. Bars = 30 μm.


Fig. S4. Effect of several hydrolytic enzymes on pneumococcal biofilms.

A–C. Destruction of R6 biofilms. (A) control, (B) treatment with chitosanase (1 unit ml−1), (C) treatment with chitinase (0.1 unit ml−1).

D. Disaggregating effect of pullulanase.

E–G. R6 biofilms were formed in the presence of different amounts of the pneumococcal LytB glucosaminidase. Growth (open bars) and biofilm formation (solid bars) are shown (E). Untreated (F) or LytB-treated (10 μg ml−1; G) were stained to test for viability using the BacLight LIVE/DEAD kit.

H–L. R6B (lytB) biofilms formed in the presence of different amounts of purified LytB (H). Biofilms formed by the strains R6 (lytB +) (I) and R6B (J) are shown, as are those formed by the R6B strain incubated with 0.5 μg ml−1 (K) or 5 μg ml−1 (L).


Fig. S5. The LytC muramidase (accession number 2WW5) was visualized using FISTGLANCE in JMOL software ( The space-filling models show cationic (blue) and anionic (red) residues. Medium blue spheres correspond to His residues. (A) general view of the protein showing its characteristic hook-like folding. The ellipse encloses the choline-binding domain. In (B), the protein has been rotated to show the catalytic pocket of LytC. (C) shows a close-up of the catalytic module (rectangle) containing the active residues Asp-273 and Glu-365 (Pérez- Dorado et al., 2010) as well as some of the basic amino acid residues proposed to interact with DNA (Arg-408, Arg-414, Lys-416 and, possibly, Arg-375).


Table S1. Linkage types in the alkali-insoluble biofilm fraction appropriate (AI-B) of isolated polysaccharide EPS of S. pneumoniae R6 biofilms, as determined by methylation analysis.


Appendix S1. Supplemental experimental procedures.

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