Mutanolysin

Axenic cultures (5 ml) of each strain were grown to OD_600nm = 0.6–0.7, harvested and resuspended in 7H9 (188 μl). Lysozyme (Roche) and Mutanolysin (Sigma) were added in 10-fold increasing concentrations (5–500 μg/ml Lysozyme and 0.5 – 50U Mutanolysin) to a final volume of 250 μl, incubated at 37°C for 120 min. Cells were centrifuged and resuspended in 7H9 (100 μl) and labelled with alk-DADA (2.5 μl) at 37°C for 60 min. Cells were centrifuged, washed in 1X PBS (Lonza) and fixed overnight in 2.5% glutaraldehyde (250 μl) at room temperature. Fluorescence conjugation for incorporated alk-DADA was performed by CuACC as previously described.

Peptidoglycan analysis was done as described earlier (Glauner et al., 1988 (link)). Essentially, the sacculi were digested with 10 U mutanolysin (Sigma-Aldrich, St. Louis, MO, United States) at 37°C in 25 mM Tris–HCl (pH 8.0) for 16 h. mutanolysin hydrolyzes β-1→4 glycosidic bond between MurNAc and GlcNAc residues in PG to form soluble muropeptide fragments. After centrifugation at 30,000 × g for 15 min, soluble muropeptide fragments in supernatant fraction were collected and reduced with 1 mg of sodium borohydride in 50 mM sodium borate buffer (pH 9.0) for 30 min and excess borohydride was destroyed by adding 20% phosphoric acid. pH was adjusted to 3–4 and the samples were loaded onto a reverse-phase C18 column (Zorbax 300 SB; 250 mm × 4.6 mm, 5 mm) connected to Agilent technologies RRLC 1200 system. Column temperature was 55°C and binding was done at a flow rate of 0.5 ml/min with 1% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA) for 10 min. Muropeptides were eluted in a gradient of 1–10% acetonitrile containing 0.1% TFA at a flow rate of 0.5 ml/min for the next 60 min (using RRLC online software called Chemstation). The absorbance of muropeptides was detected at 205 nm.

A culture volume of 2 mL was used for the extraction of gDNA, using a GeneJET Genomic DNA Purification kit (K0721, ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol for Gram-positive bacteria, but with minor modifications [28 (link)], including the addition of 20 U/mL mutanolysin (Sigma-Aldrich, St Louis, MO, USA) and 30 mg/mL lysozyme (Sigma-Aldrich, St Louis, MO, USA) in the lysis buffer and an additional incubation (37 °C for 60 min) with 2.0 µg/mL DNase-free RNase (Sigma-Aldrich, St Louis, MO, USA). The concentration (absorbance at 260 nm) and purity (absorbance ratio, 260/280 nm) of the extracted gDNA were estimated by using a Nanodrop ND 1000 spectrophotometer (ThermoFisher Scientific, MA, USA), and the gDNA was stored in aliquots at −20 °C until further use.

Cells were harvested and DNA was extracted as previously described by Siebert et al. [24 (link)]. In brief, 30 mL bovine raw milk were treated with 1.8 mL 0.3 M EDTA. After the cell harvesting by centrifugation (20 min at 4 °C) and the removal of the milk fat and skimmed milk in the supernatant, the selective lysis of the somatic DNA was performed using proteinase K (20 mg/mL, AppliChem GmbH, Darmstadt, Germany) and DNase I (Thermo Fisher Scientific, Waltham, MA, USA). The DNA extraction was performed with the PowerFood Microbial DNA isolation kit (Qiagen, Hilden, Germany), modified by an additional enzymatic lysis step. Towards this end, lysozyme (25 µg/mL, Carl Roth) and mutanolysin (100 U, Sigma-Aldrich, St. Louis, MO, USA) were added to the cell suspensions together with the MBL solution of the DNA isolation kit, followed by an incubation at 37 °C and 350 rpm for 30 min. After an additional treatment with proteinase K (12.5 mg/mL, AppliChem), the remaining bacterial cells were disrupted in tubes with silica beads using a FastPrep-24TM instrument (MP Biomedicals, LLC, Irvine, CA, USA). The subsequent DNA isolation followed the manufacturer’s protocol, i.e., that of the PowerFood Microbial DNA isolation kit. The DNA was finally eluted in 2 × 24 µL of PCR-grade water (preheated to 55 °C).

Bacterial cells were harvested from 40-ml overnight broth cultures by centrifugation (3,000 × g, 30 min) and lysed and treated as follows. (i) Nonpneumococcal cells were suspended in 1-ml lysis buffer (0.1 M NaCl, 0.05 M HEPES, 1 mM CaCl₂, 1 mM MgCl₂ [pH 7.5]) containing 100 U of mutanolysin (Sigma) and 1 mg lysozyme (Sigma) and incubated at 37°C for several hours until more than 95% of the cells were digested as evaluated by Gram staining. (ii) Pneumococcal cells were suspended in 1 ml 0.1% sodium deoxycholate in PBS. This lysis buffer activates the autolysin and induces complete lysis of the pneumococcal cells. Cell debris was removed from the bacterial extracts by centrifugation (10,000 × g, 30 min), and proteins in the supernatants were digested by adding proteinase K (10 µg/ml) for 2 h at 50°C. The protease activity was finally blocked by adding 15 µl stock solution of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF [with 17.4 mg/ml isopropanol]) per ml of extract to a final concentration of 1.5 mM. A 0.01% solution (1 mg per 10 ml of saline) of purified pneumococcal C-polysaccharide (Statens Serum Institut) was used as a control.

Fresh fecal samples were collected in a plastic tube containing RNAprotect Bacteria Reagent (Qiagen). The samples were centrifuged at 5000 g for 10 min, and the pellet was frozen immediately at −80 °C until use. Bacterial RNA extraction was performed as described previously with a few modifications52 (link). Briefly, the pellet was suspended and homogenized in 200 μl Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) containing 30 mg/ml lysozyme (SIGMA-ALDRICH, Tokyo, Japan), 5000 U/ml mutanolysin (SIGMA-ALDRICH) and 20 mg/ml proteinase K (Qiagen). The samples were incubated with gentle shaking for 60 min at room temperature, and 700 μl of RLT buffer (Qiagen) was added to the lysates. Total RNA was purified using RNeasy Mini kit (Qiagen) with an on-column DNase treatment following the manufacturer’s protocol.