The N-Acetylglucosaminidase LytB of Streptococcus pneumoniae Is Involved in the Structure and Formation of Biofilms

It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

ABSTRACT

The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, ΔlytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytB_E585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either ΔlytB or producing the inactive LytB_E585A) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 μg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytB_E585A mutant formed biofilm more rapidly than ΔlytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed.

IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

INTRODUCTION

Streptococcus pneumoniae (pneumococcus) causes community-acquired pneumonia, meningitis, and sepsis and is commonly responsible for middle-ear infections in children. Associated with substantial morbidity and mortality, the pathogen generates considerable health care costs (1, 2). Pneumococcus is carried asymptomatically in the nasopharynx of <10% of healthy adults but in as many as 25 to 65% of children, where colonization begins shortly after birth (3). Pneumococcal biofilms appear on the epithelium of the adenoids and mucosae in children, leading to recurrent middle-ear infections and otitis media (OM) with effusion, and are found on the sinus mucosa of patients with chronic rhinosinusitis (4) and can also form in vitro (5). Biofilm formation is an efficient way of evading both the classical and PspC-dependent complement pathways of the host immune system (6). Extracellular DNA (eDNA) secures the structural stability of S. pneumoniae biofilms and thus plays an important role in their development (7–10). Indeed, eDNA appears to be an abundant component of in vitro S. pneumoniae biofilms (11), forming intercellular complexes with different proteins (12, 13) including LytB, an endo-β-N-acetylglucosaminidase (EC 3.2.1.-) (14). LytB has a modular structure comprising an N-terminal choline-binding domain (consisting of 18 choline-binding repeats [CBRs]) and a C-terminal domain belonging to the GH73 family of glycoside hydrolases (15). Through interaction with the eukaryotic-like serine/threonine kinase StkP (16), LytB is responsible for daughter cell separation (17) but is also involved in pneumococcal attachment to nasopharyngeal and human pleural mesothelial cells (18, 19), as well as in adhesion to and invasion of lung epithelial cells (20). LytB is also required for optimal in vitro biofilm formation (7), and the addition of purified exogenous LytB has been reported to boost biofilm formation in a ΔlytB mutant (11). LytB is therefore a promising target for the development of a universal pneumococcal vaccine; indeed, an anti-LytB antiserum has been shown to significantly protect mice from lethal challenge with different pneumococcal strains (21). More recently, it has been shown that immunization with LytB activates complement-mediated phagocytosis and induces protection against pneumonia and sepsis (22). Interestingly, antibodies against LytB were detected after nasopharyngeal colonization and acute OM in children (23, 24), two conditions in which biofilm formation by S. pneumoniae—alone or in conjunction with nontypeable Haemophilus influenzae—plays an essential role (25).

The present work provides evidence that LytB plays a role more essential than previously thought in the formation of pneumococcal biofilms. Moreover, LytB plays a dual role in biofilm development and the results obtained show the importance of LytB not only as a chain-dispersing enzyme (17) but also as a structural protein capable of forming complexes with eDNA in the biofilm matrix.

RESULTS

Enzymatically active LytB is essential for the development of pneumococcal biofilms.

In collaboration with the major autolysin LytA, LytB is responsible for the typical S. pneumoniae diplococcus phenotype. A direct consequence of lytB deletion is the formation of long chains of cells (17) and a reduced ability to produce in vitro biofilms (7, 11). We have now analyzed in more detail the biofilms formed not only by the previously studied ΔlytB mutant strain (R6B) but also by strain P255, a pneumococcal strain synthesizing an enzymatically inactive LytB protein (LytB_E585A). In LytB_E585A, the catalytic proton donor Glu585 is substituted by Ala, resulting in the complete loss of N-acetylglucosaminidase activity (14). As shown in Fig. 1, both mutants failed to form distinct biofilms, as determined by crystal violet (CV) staining and viable cell counting (Fig. 1a). Due to the characteristic chaining of lytB mutants, efforts were made to efficiently disperse the cells before plating (see the Materials and Methods). The biofilms formed by the R6B and P255 strains were also analyzed using confocal laser scanning microscopy (CLSM). Maximum intensity projections suggested that, although in reduced amounts, biofilms may be still formed by the lytB mutants (Fig. 1b). This conclusion, however, appears to be misleading because orthogonal projections of biofilms showed them to be very shallow (Fig. 1c), in agreement with the cell counting results (see above). A plausible explanation for this discrepancy may reside in the tangling of chains formed by lytB pneumococcal mutants. Whatever the reason, and as it occurs in the case of maximum intensity projections, attempts to quantify fluorescence images provided results that did not fit with cell-counting determinations (data not shown). These results taken together strongly suggested that LytB is of greater importance for biofilm formation than previously believed.

FIG 1.

S. pneumoniae lytB mutants fail to form substantial biofilms. (a) Biofilms formed by the R6 (lytB⁺), R6B (ΔlytB), and P255 (enzymatically inactive LytB) strains were grown for 5 h at 34°C on microtiter plates. The black and open bars represent total growth (adherent plus nonadherent cells) and the biofilm (adherent cells), respectively. Biofilm formation was quantified using CV. *, P < 0.001 compared to the biofilm formed by strain R6. Gray bars indicate viable cells in the biofilm. The results are the average of three independent experiments each performed in triplicate. (b) Maximum intensity projections were obtained in the x−y (individual scans at 1 μm intervals) and x−z (images at 5 μm intervals) planes. Biofilms were formed on glass-bottomed plates after 5 h of incubation at 34°C. Cells were stained, using the BacLight viability kit from Invitrogen, to reveal living (green fluorescing) and dead (red fluorescing) bacteria. (c) Orthogonal CLSM images of biofilms formed by the S. pneumoniae R6 strain and two lytB mutants. The images on the right and the bottom show a representative region of the x–y plane over the depth of the biofilm in both the x–z and y–z dimensions of biofilm. In all images the scale bar equals 25 μm.

Previous work has shown that the full catalytic activity of LytB is required for pneumococcal adhesion to, and invasion of, human lung epithelial cells (18). It has also been reported that exogenous LytB inhibited biofilm formation in S. pneumoniae R6 (lytB⁺) when added at concentrations of ≥5 μg/ml (11). In contrast, the addition of 0.5 to 2.0 μg/ml of LytB restored biofilm formation in R6B (ΔlytB). Further evidence that enzymatically active LytB was required for biofilm formation was obtained using LytB_E585A. Indeed, this protein was unable either to inhibit biofilm formation (in R6) or to restore it (in R6B or P255) when used in the range of 0.01 to 20 μg/ml (data not shown). Additional support for the importance of the enzymatic activity of LytB in biofilm formation was obtained when deletion forms of LytB were tested for reestablishing biofilm formation (Fig. 2). As mentioned above, LytB from R6 has 18 CBRs. Rico-Lastres et al. demonstrated that a minimum of 4 CBRs are required for detectable enzymatic activity (as determined by chain-dispersing capacity) (14). In parallel with these results, we have observed that only deleted forms of LytB containing ≥8 CBRs (GFP-LytBΔ2 and GFP-LytBΔ3) restored biofilm formation by R6B to a significant extent (P < 0.001) (Fig. 2).

FIG 2.

Biofilm-forming capacity of R6B (ΔlytB) in the presence of LytB and LytB deletion variants. R6B biofilms were formed after 5 h of incubation at 34°C on polystyrene microtiter plates in the presence of 0.5 μg/ml of either LytB or deleted LytB constructs. The black and open bars represent total growth (adherent plus nonadherent cells) and the biofilm (adherent cells), respectively. Biofilm formation was quantified using CV. *, P < 0.001 compared to the biofilm formed by strain R6B (control). The results are the average of three independent experiments each performed in triplicate.

Kinetics of biofilm development.

We examined the kinetics of biofilm formation for both lytB mutants (R6B and P255) in the presence of 0.5 μg/ml externally added LytB (Fig. 3). In the presence of the added enzyme, the biofilms produced were compact and thick, and the typical chains, characteristic of lytB mutants, disappeared as incubation proceeded. At 5 h of incubation, the biofilms formed by the mutants were virtually indistinguishable from that formed by the wild-type strain R6 (see Fig. 1b for comparison). Additional experiments showed that this externally added LytB was evenly distributed throughout the biofilm (Fig. S1 in the supplemental material). Although mutants regained the wild-type phenotype, the P255 strain formed a more compact and thicker biofilm than the ΔlytB mutant, and apparently did so more quickly (Fig. 3) and at a lower enzyme concentration (Fig. S2). In addition, compared to the deletion mutant, biofilm formation in the LytB-defective P255 strain was less susceptible to the inhibitory effect of the enzyme when used at high concentrations (Fig. S2). When LytB was added at concentrations of ≥5 μg/ml, the biofilm-forming capacity of P255 was superior to that of a control without LytB.

FIG 3.

Kinetics of biofilm development of lytB mutants by CLSM. Development of the R6B (ΔlytB) biofilm without treatment (untreated control) and of R6B and P255 (enzymatically inactive LytB) strains, both incubated with 0.5 μg/ml of LytB for 2 to 5 h at 34°C. After treatment, the cells in the biofilms were stained with SYTO 9. Maximum intensity projections were obtained in the x−y (individual scans at 0.5 μm intervals) and x−z (images at 5 μm intervals) planes. The images are representative of experiments performed in triplicate. In all images the scale bar equals 25 μm.

Extracellular DNA and LytB are crucial for optimal biofilm formation.

Together with LytB, eDNA is also important in both biofilm formation and maintenance (7). The latter also applies to the thin biofilms formed by the lytB mutants (Fig. 4). Inhibition assays (in which DNase I was added before biofilm formation) and dispersal tests (in which DNase I was added to a preformed biofilm) revealed a significant reduction (P < 0.001) in the biofilm of both mutants compared to the corresponding untreated control. Moreover, R6B biofilms appeared to be more susceptible (P < 0.01) to the dispersing activity of DNase I than those formed by strain P255 (Fig. 4a). This suggested that in P255 biofilms, eDNA was partly protected from enzymatic degradation, possibly because it was forming LytB_E585A–DNA complexes in the biofilm matrix. Experimental support for this proposal was obtained by DNase I treatment of LytB-DNA complexes formed in vitro (12) (Fig. 4b). Complexed DNA was resistant to DNase I treatment (as shown by agarose gel electrophoresis), whereas naked plasmid DNA (pGL30) was completely hydrolyzed. Interestingly, LytB-bound DNA appears to remain intact since it migrates to its expected position in gels after its removal from the protein–DNA complex with proteinase K. In contrast with the important role of eDNA in biofilm formation and maintenance, RNase treatment of pneumococcal biofilms showed no detectable effect (data not shown). This result was in agreement with previous evidence showing that, with only a few exceptions (26–28), extracellular RNA seems not to be a critical biofilm element (5).

FIG 4.

Inhibition and dispersal of lytB biofilms with DNase I, and inhibition of DNA degradation by LytB–DNA complex formation. (a) The indicated S. pneumoniae strains were grown overnight at 37°C to an A₅₅₀ of ∼0.5 (corresponding to the late exponential phase of growth) in C+Y medium, centrifuged, and adjusted to an A₅₅₀ of 0.6 with fresh medium. The cell suspensions were then diluted 100-fold and 200 μl aliquots distributed into the wells of microtiter plates, which were then incubated for 5 h at 34°C (open bars). Other samples received DNase I (100 μg/ml) (hatched bars) and were incubated as above (inhibition assay). For dispersal assays (gray bars), after biofilm development (4 h at 34°C), DNase I was added at 100 μg/ml, and incubation allowed to proceed for an additional 1 h at 34°C. Biofilm formation was always measured using CV staining. In all assays, black bars indicate growth (adherent plus nonadherent cells). *, P < 0.001 compared with the corresponding untreated control; #, P < 0.01 in inhibition assays where lytB mutants were compared to each other. The results are the average of four independent experiments each performed in triplicate. (b) LytB (1 μg) and pGL30 (40 ng) were incubated at 37°C for 50 min in 10 mm sodium phosphate buffer (pH 6.0) containing 1 mM MnCl₂ (final volume, 15 μl). Lanes labeled pGL30 or DNase I correspond to the undigested plasmid and DNase I-digested (50 μg ml⁻¹; 20 min, 37°C) pGL30, respectively. LytB, undigested LytB–pGL30 complex. The binding mixture was also digested with either DNase I or proteinase K (100 μg ml⁻¹; 15 min, 37°C) after allowing the formation of the LytB–DNA complex. The different samples were then analyzed by agarose gel (0.7%) electrophoresis. (c) Visualization of eDNA in an early biofilm (3 h at 34°C) of S. pneumoniae R6B by CLSM. A combination of SYTO 59 (red; pneumococci) and anti-dsDNA antibody, followed by Alexa Fluor-488 goat anti-mouse IgG (green; eDNA) was employed for biofilm staining. Maximum intensity projections were obtained in the x−y (individual scans at 0.5 μm intervals) and x−z (images at 5 μm intervals) planes. Scale bars equal 25 μm.

Additional evidence for the presence of eDNA in the extracellular matrix of a lytB biofilm was obtained by CLSM. Staining of early R6B biofilms (after 3 h growth) with anti-double-stranded DNA (dsDNA)-Alexa 488 revealed eDNA to be distributed throughout (Fig. 4c). Long eDNA filaments and condensed eDNA spots, both containing attached bacteria, were evident. Of note, when either homologous or heterologous DNA (e.g., from salmon sperm) was added to pneumococcal cultures of any of the two lytB mutants, biofilm formation was not significantly affected (not shown), in agreement with previous results using strain R6 (lytB⁺) (7).

The distribution and appearance of eDNA over time in the biofilms of both lytB mutants (R6B and P255) in the presence of 0.5 μg/ml of LytB were then examined (Fig. 5). At the earliest time point (2 h), eDNA fibers were abundant in the biofilm of P255, whereas aggregated or condensed eDNA was seen in the R6B biofilm (Fig. 5b). Previous experiments have demonstrated the in vitro formation of large aggregates of LytB-DNA along with aggregates with apparent protein-free DNA fibers (12). After 4 h, eDNA fibers with bound bacteria were seen at the surface of the R6B biofilm, while condensed eDNA appeared at the bottom, although this was difficult to perceive after 5 h of incubation (Fig. 5c). Condensed eDNA may represent a late step in the formation of LytB−DNA complexes. That these complexes were also effective as inducers of R6B biofilm formation suggests that LytB still conserves some N-acetylglucosaminidase activity when complexed with eDNA (Fig. S3).

FIG 5.

Visualization of eDNA in the biofilm of S. pneumoniae R6B (ΔlytB) during biofilm formation by lytB mutants. (a) Kinetics of the biofilms of R6B and P255 (enzymatically inactive LytB) incubated for 2 to 5 h with LytB (0.5 μg/ml). Biofilms were stained with a combination of SYTO 59 (red) and anti-dsDNA antibody, followed by Alexa Fluor-488 goat anti-mouse IgG (green). (b) A detailed view of eDNA in the biofilms of S. pneumoniae R6B and P255 grown for 2 h at 34°C in the presence of 0.5 μg/ml of LytB (see panel a). (c) Distribution of eDNA in biofilms of S. pneumoniae R6B. CLSM images of R6B biofilms incubated for 4 h and 5 h at 34°C in the presence of 0.5 μg/ml LytB. Extracellular DNA and the bacteria of the biofilms were stained as described for panel a. Maximum intensity projections were obtained in the x−y (individual scans at 0.5 μm intervals) and x−z (images at 6 μm intervals) planes. In all images, scale bars equal 25 μm.

DISCUSSION

LytB appears not to be required for planktonic growth in vitro, (7, 16, 17, 29, 30). However, in the present study, it is shown that there appears to be a different case for biofilm formation. Different studies have shown that in vitro biofilm formation by S. pneumoniae is a multigenic trait, and that the mutation of just one gene might lead to a partial inhibition of biofilm production (7, 31–35). However, we show here that the inactivation of lytB alone caused the near complete loss of in vitro biofilm-forming capacity, which contrasts with previous results (7). It is tempting to speculate that chain formation by itself is the determining factor in the inability to form biofilms, although it has been reported that several streptococci closely related to S. pneumoniae, and which grow in long chains, form biofilms virtually identical to that of a pneumococcal LytB⁺ strain (7). Of note, Enterococcus faecalis and Streptococcus gordonii mutants lacking, respectively, the AtlA or LytB N-acetylglucosaminidases also formed chains of cells and, as in the present case, were impaired in biofilm formation (36, 37). Although chain formation is not uncommon during biofilm formation by rod-shaped bacteria (38, 39), it is still unclear whether chain-forming mutants show impaired biofilm formation. Surface attachment is the first step in biofilm formation and it has been reported that, at least in Escherichia coli and Pseudomonas aeruginosa, it takes place at the cell poles (40), in agreement with the polar localization of several surface proteins with adhesive properties (41). In certain Gram-positive bacteria with low G+C content, several surface proteins involved in pathogenesis (particularly those involved in cell wall metabolism) show a septal/polar localization (42). If this were the case for S. pneumoniae, initial attachment to the substrate would be impaired when the cocci grow in chains. It should be remembered that LytB actually localizes at the cell poles (17), and the lack of this protein (as in the R6B deletion mutant) would alter the first step of biofilm formation, i.e., the binding of cells to the substrate. In the enzymatically inactive LytB mutant (strain P255), substrate attachment might be impaired by steric hindrance because of the chaining phenotype of the mutant. Nevertheless, the presence of LytB_E585A at the poles appears to allow a rapid recovery of the biofilm-forming capacity of P255 when the purified wild-type enzyme is externally added.

Extracellular DNA and polysaccharide(s) are essential components of the matrix of in vitro S. pneumoniae biofilms, where eDNA is at least partly complexed with several CBPs, including LytB (11, 12). It has been previously reported that Atl, the principal autolysin of E. faecalis, is critical for eDNA release and biofilm development (37). This contrasts with the case of LytB (which lacks any detectable autolytic activity), as abundant eDNA was detected in R6B early biofilms (Fig. 4c), possibly as a consequence of a partial lysis of the culture caused by LytA and LytC S. pneumoniae autolysins.

The possible involvement of LytB in the synthesis of matrix polysaccharide warrants some comment. The extracellular β-polysaccharide, unlike that found in the capsule, is composed of residues of Glcp(1→4) and GlcNAc(1→4) (in their deacetylated forms) (11). Rico-Lastres et al. (14) recently noted that LytB, in addition to its hydrolytic activity, shows a small amount of glycosyltransferase activity that incorporates GlcNAc units into chitotetraose/chitopentose substrates. Interestingly, since the pneumococcal matrix polysaccharide is highly susceptible to the action of chitinases (11), a role for LytB in the synthesis of the extracellular polysaccharide might be proposed. Unfortunately, the weakness of the biofilms formed by the lytB mutants precluded investigations using the calcofluor stain and/or appropriate lectins (11) to determine whether matrix polysaccharide is absent from them.

As mentioned above, the nasopharyngeal microbiome is primarily associated with the high abundance of S. pneumoniae, H. influenzae, and Moraxella catarrhalis during OM (43). These microorganisms usually aggregate and grow as biofilms (44), which provides an increased resistance to host defense mechanisms (6). Previous studies using animal models have documented that LytB plays a significant role in nasopharyngeal colonization (18, 45), which is the first step in pathogenesis leading to local (OM and sinusitis) and severe (pneumonia, sepsis, and meningitis) invasive pneumococcal infections (46). More recently, a rise in the serum levels of specific IgG anti-LytB antibodies was reported in children who experienced S. pneumoniae nasopharyngeal colonization and OM (23). This is very important because higher levels of anti-LytB antibodies are associated with a reduced frequency of OM in children, as recently reported (24). Unfortunately, the real role of LytB in the colonization of the nasopharynx (i.e., biofilm formation) was largely unknown, probably because the lytB gene appears to be under the control of a complex mix of environmental factors, including major global responses to nutrient limitation and other stresses (47–51). Microarray analyses have also shown lytB to be upregulated upon contact of the bacterium with the pharyngeal epithelium (52) or when pneumococcal biofilms are dispersed by incubation with influenza A virus (53), although, to the best of our knowledge, quantification of the LytB protein has never been reported.

The present in vitro study shows that S. pneumoniae LytB is essential for biofilm formation, with this protein playing an active role in the separation of daughter cells, which then form the nucleation points for the development of microcolonies, and, eventually, biofilms. Moreover, LytB is apparently not involved in eDNA release. In addition, and regardless of its enzymatic activity, LytB has a structural and/or adhesive role in S. pneumoniae biofilms, as deduced from the formation of the compact biofilm when a defective mutant (P255) was rescued by adding external LytB. The capacity of LytB to form complexes with eDNA probably helps impose the architecture of the biofilms formed. In addition to its peptidoglycan hydrolytic activity, LytB may also be involved in the synthesis of the exopolysaccharide component of the matrix of pneumococcal biofilms.

MATERIALS AND METHODS

Strains, media, and growth conditions.

Pneumococcal strains R6 (54), R6B (26), and P255 were grown in pH 8-adjusted C medium (CpH8) supplemented (or not, as required) with 0.08% yeast extract (C+Y) medium (7). Cells were incubated at 37°C without shaking and growth was monitored by measuring absorbance at 550 nm (A₅₅₀). The procedure followed to transform S. pneumoniae is detailed elsewhere (7).

Strain P255 was constructed using plasmid pRGR5E585A (14), kindly supplied by M. Menéndez. Initial transformation experiments using this plasmid (as the donor) and the R6 strain (as recipient) were unsuccessful, probably because there was no direct way to select for the transformants, or because the transformation efficiency was insufficient. We therefore took advantage of the fact that naturally competent bacteria are cotransformed by unlinked markers at unexpectedly high frequencies (“congression”) (55, 56). The following two-step procedure provided the best results. First, cells of the highly competent strain M32 (ΔlytA32) (57) were simultaneously transformed with pRGR5E585A and chromosomal DNA from strain M22 (streptomycin-resistant; Str^r) (58). Several Str^r transformants were picked out, incubated in C+Y medium at 37°C until the stationary phase of growth, and then visually inspected for chain formation. Since the recipient strain (M32) is a lytA deletion mutant, it performs no lysis in the stationary phase of growth, allowing for the long term incubation of the transformants. This is optimal for chains to develop and sediment out at the bottom of the tube (59). An Str^r clone forming long chains of cells (strain P253) was selected. DNA sequencing showed that P253 contained the desired lytB mutation coding for LytB_E585A. Competent cells of strain R6 were then simultaneously transformed with an 1,848 bp-long lytB_P253 fragment (prepared by PCR amplification of strain P253 DNA with oligonucleotides lytB-UP [5′-GAATGGGTAGAAGACAAGGGAG-3′] and lytB-DN [5′-CTAATCTTTGCCACCTAGCTTCTC-3′]), and P233 chromosomal DNA. P233, a novobiocin-resistant (Nov^r) strain, has been previously described (60). One of the Nov^r transformants growing in long chains was purified by three consecutive passages on plates and named P255. The accuracy of the construct was assessed by nucleotide sequencing of the lytB_P255 allele. Since LytB contains a 23-amino-acid-long, cleavable signal peptide (29), Glu585 corresponds to Glu608 in the unprocessed form of the enzyme of the R6 strain. It is worthy of note that the transcription start site of the lytB gene had not been determined until recently (61).

Biofilm formation was determined by the ability of cells to adhere to the walls and base of 96-well, flat-bottomed polystyrene microtiter dishes (Costar 3595; Corning Inc.) using a modified version of a previously reported protocol (32). Unless stated otherwise, cells were grown in C+Y medium to an A₅₅₀ of ∼0.5 to 0.6, sedimented by centrifugation, resuspended in an equal volume of the indicated prewarmed medium, diluted 1/10 or 1/100, and then dispensed at a volume of 200 μl per well. Plates were incubated at 34°C for 2 to 5 h and bacterial growth (planktonic plus biofilm-forming cells) was determined by measuring the A₅₉₅ using a VersaMax microplate absorbance reader (Molecular Devices). The biofilm was stained with 1% CV, after which the plates were incubated for 15 min at room temperature and then rinsed three times with distilled water to remove nonadherent bacteria. After solubilizing the biofilm in 95% ethanol (200 μl per well), the A₅₉₅ was determined using a VersaMax microplate absorbance reader. Biofilm disaggregation was performed by gentle pipetting and slow vortexing (11) until 80 to 90% of the cells were diplococci or short chains (≤5 cells), as verified using phase-contrast microscopy. Quantification of viable cells (planktonic and biofilm cells) was performed in blood agar plates after overnight incubation at 37°C.

LytB, deleted forms of LytB, and LytB_E585A were purified, and LytB–DNA complexes prepared, as previously described (12, 14). The deleted forms of LytB contained a green fluorescent protein (GFP) fused to the N terminus of LytB (GFP-LytB) or truncated LytB mutants with 13 (GFP-LytBΔ2), 8 (GFP-LytBΔ3), 4 (GFP-LytBΔ4), or no CBRs (LytB_CAT). These proteins were kindly provided by P. García. It has been reported that the N-terminal GFP tag did not interfere with the catalytic activity (14). Unless stated otherwise, a mixture of plasmid pGL30 (100 ng) and LytB (2.5 μg) was incubated in 10 mM sodium phosphate buffer (pH 6.0) containing 1 mM MnCl₂ (final volume, 15 μl) for 1 h at 37°C. The mixture was then added to an R6B culture (LytB final concentration 0.5 μg/ml), and incubated under biofilm-forming conditions. The fusion protein GFP-LytB was purified as described elsewhere (17).

Microscopic observation of biofilms.

For the observation of S. pneumoniae biofilms by CLSM, pneumococcal strains were grown on glass-bottomed dishes (WillCo-dish) for 2 to 5 h at 34°C as previously described (11). Following incubation, the culture medium was removed and the biofilm rinsed with phosphate-buffered saline (PBS) to remove nonadherent bacteria. The biofilms were then stained with anti-dsDNA antibody (ab27156, Abcam) (at 2 to 25 μg/ml each) and/or SYTO 59 (10 μM) (S11341, Invitrogen). All staining procedures involved incubation for 10 to 20 min at room temperature in the dark, except when incubating with mouse anti-dsDNA antibody (2 μg/ml); this involved a fixation step at room temperature with 3% paraformaldehyde for 10 min. The biofilms were then rinsed with 0.5 ml PBS and incubated for 1 h at 4 °C followed by a 30-min incubation at room temperature in the dark with Alexa fluor 488-labeled goat anti-mouse IgG (1:500) (A-11001, Invitrogen) (diluted 1/500). Biofilms were also stained with the bacterial viability BacLight kit (Invitrogen, Thermo Fisher Scientific), showing viable (green fluorescence) and nonviable (red fluorescence) bacteria. After staining, they were gently rinsed with 0.5 ml PBS. Observations were made using a Leica TCS-SP2-AOBS (Acousto-Optical Beam Splitter)-UV CLSM (Mannheim, Germany) with objective HCX PL APO CS 63×/1.4 oil immersion and zoom 2. Laser lines at 488 nm (for excitation of GFP, SYTO 9, and Alexa fluor-488) and 561 nm (for excitation of SYTO 59 and propidium iodide) were provided by an argon laser and a DiodeP solid state laser, respectively. Detection ranges were set to eliminate cross talk between fluorophores. Emission ranges were 500 to 546 nm (for SYTO 9, GFP, and Alexa fluor-488) and 617 to 666 (for SYTO 59 and propidium iodide). The image resolution was 8 bits and format 512 × 512 pixels. Laser intensity and gain were kept the same for all images and they were analyzed using LCS software from Leica. Maximum intensity projections were obtained in the x–y (individual scans at 0.5 to 1 μm intervals) and x–z (images at 5 to 6 μm intervals) planes. Orthogonal projections were also obtained.

Statistical analysis.

Data comparisons were performed using a two-tailed Student’s t test.