The procedure used has been described elsewhere [30 (link), 36 (link)]. The PG (200 μg) was digested with 20 μg of mutanolysin from Streptomyces globisporus (Sigma) for 18 h at 37°C in 12.5 mM sodium phosphate buffer (pH 5.8). The enzyme reaction was stopped by boiling the sample for 3 min, the digested PG was mixed with sodium borohydride (2 mg) for 15 min. The pH of the samples was then adjusted to 2.0, and the samples were centrifuged to remove insoluble material. We used a linear gradient from 50 mM sodium phosphate buffer (pH 4.3) to 50 mM sodium phosphate buffer (pH 5.1) containing 15% methanol for 120 min on a Hypersil ODS column (4.6 × 250 nm; 5 μm particles; ThermoHypersil-Keystone) at 52°C using a flow rate of 0.5 ml/min. UV detection was carried out at 205 nm. The quantity of each muropeptides was assessed by measuring the area of the corresponding peak. We only compared PGs that were extracted simultaneously. For assessing evolution of the PG structure in the different phyla, we determined for each species the total ratio of GM4/GM5 that is calculated as followed (GM4+GM4_GM4)/(GM5+GM5_GM4) where GM4_GM4 and GM5_GM4 represent dimers.
Mutanolysin from streptomyces globisporus
Manufactured by Merck Group
Sourced in United States
Mutanolysin is an enzyme derived from the bacterium Streptomyces globisporus. It is a muramidase that cleaves the glycosidic bonds in peptidoglycan, a major component of bacterial cell walls. This enzymatic activity can be used to facilitate the extraction and analysis of cellular components from Gram-positive bacteria.
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Lab products found in correlation
Gemini c18 column, by Phenomenex (1 mentions)
Microtof 2 mass spectrometer, by Bruker (1 mentions)
Ultimate 3000 rs hplc system, by Thermo Fisher Scientific (1 mentions)
Speedvac vacuum centrifuge, by Thermo Fisher Scientific (1 mentions)
Mutanolysin, by Merck Group (1 mentions)
Lysozyme from chicken egg white, by Merck Group (1 mentions)
Taq dna polymerase, by Thomas Scientific (1 mentions)
5 protocols using mutanolysin from streptomyces globisporus
1
Peptidoglycan structure analysis
2
Peptidoglycan Digestion and Analysis
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PGN (0.5 mg) was resuspended in 70 μl of sodium phosphate buffer (200 mM, pH 6.0) and digested with mutanolysin from Streptomyces globisporus (50 μg ml− 1; Sigma). After incubation over night at 37 °C under constant shaking, the reaction was stopped by heating at 100 °C for 25 min. To produce single peptides, PGN was further digested with 50 μg ml− 1 of N-acetylglucosaminidase NagZ [42 (link)] for 6 h and 100 μg ml− 1 of amidase AmiE [42 (link)] overnight. Muropeptides were reduced by mixing 100 μl of the digest with 100 μl sodium borate buffer (0.5 M, pH 9.0) and adding 5 mg of sodium borohydride. After incubation for 30 min at room temperature, the reaction was stopped with 5–10 μl 20% phosphoric acid, adjusting the pH to 3.5. After centrifugation (12,000 g, 10 min, room temperature), the supernatant was dried in a Speed Vac vacuum centrifuge (Thermo Fisher Scientific) and dissolved in 50 μl of distilled water. Preparation aliquots of 5 μl were applied to HPLC at a flow rate of 0.2 ml min− 1 and an elution profile (using buffer A: formic acid with 0.05% ammonium formate, and buffer B: 100% acetonitrile) as described previously [44 (link)]. LC-ESI-MS measurements were performed using a Gemini C18 column (150 × 4.6 mm, 110 Å, 5 μm; Phenomenex) and an UltiMate 3000 RS HPLC system (Dionex) coupled to a MicrO-TOF II mass spectrometer (Bruker), operated in positive ion mode.
3
Peptidoglycan Characterization of Lactobacillus
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PG from L. plantarum strains was prepared by treating a bacterial pellet with SDS, nucleases, and proteases solutions in order to eliminate all the cellular components except PG, according to a protocol previously described (Courtin et al., 2006 (link)). This protocol was slightly modified by applying DNase (50 μg ml−1) and RNase (50 μg ml−1) treatments before hydrofluoric acid extraction. PG was digested with mutanolysin from Streptomyces globisporus (Sigma-Aldrich). The resulting muropeptides were analyzed by RP-HPLC as previously reported (Courtin et al., 2006 (link)). Muropeptides were identified according to their retention times by comparison to the previously published reference chromatogram for L. plantarum PG (Bernard et al., 2011a (link)). In addition, disaccharide-dipeptide (Di) purified from Lactobacillus casei (Regulski et al., 2012 (link)) was used as standard. The relative abundance (in %) of Di (with or without O-acetylation) was calculated as the ratio of the areas of the two peaks over the sum of the areas of all the identified peaks on the chromatogram.
4
Peptidoglycan Extraction and Muropeptide Analysis
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Bacteria (E. faecium and E. faecalis) were grown in fresh BHI medium with shaking at 37°C to log-phase (OD600 of 0.6). Peptidoglycan was extracted by resuspending the bacterial cell pellet in 0.25% SDS solution in 0.1 M Tris-HCl, pH 6.8 and boiling the suspension for 20 min at 100°C in a heating block as previously described (Kühner et al., 2014 (link)). The resulting insoluble cell wall preparation was washed with distilled water six times until free of SDS. The cell wall was purified by treatment with benzonase followed by trypsin digestion. Then, insoluble cell wall was recovered by centrifugation (16,000 x g, 10 min, 4°C), and washed once in distilled water. To obtain pure peptidoglycan, cell wall was then suspended in 1 M HCl and incubated for 4 hr at 37°C in a shaker to remove wall teichoic acid. The insoluble material was collected by centrifugation (16,000 x g, 10 min, 4°C) and washed with distilled water repeatedly until the pH was 5–6. The final peptidoglycan was lyophilized and stored at −20°C. For muropeptide analysis, purified peptidoglycan was digested with mutanolysin from Streptomyces globisporus (Sigma, 10 KU/ml of mutanolysin in ddH2O) in 10 mM sodium phosphate buffer, pH 4.9 for 16 hr at 37°C. The enzyme reaction was stopped by incubating at 100°C for 3 min. The resulting soluble muropeptide mixture was then analyzed by ANTS labeling described below.
5
Molecular Cloning and Sequencing
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All restrictions enzymes were purchased from New England Biolabs (NEB, USA), whereas the lysozyme from chicken egg white and mutanolysin from Streptomyces globisporus were purchased from Sigma-Aldrich (Sigma-Aldrich, USA). Phusion polymerase (NEB, USA) was used to generate PCR amplicons for Sanger sequencing, whereas Taq DNA polymerase (Denville Scientific, USA) was used for screening purposes. The oligonucleotides used in this study are listed on Additional file 5 : Table S2 (Integrated DNA Technology-IDT, USA).
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