Lysozyme

Lysates of overnight L. monocytogenes cultures of Serbia-2019 were obtained as previously described, with modifications [27 ]. Then, 100 µL of a 24-h culture grown in heart–brain broth (Difco, Detroit, MI, USA) at a temperature of 37 °C was resuspended in a pre-lysis buffer of the following composition: 20 mM Tris (hydroxymethyl) aminomethane (Tris)/hydrochloric acid (HCl); 2 mM EDTA ethylenediaminetetraacetic acid (EDTA); 1% Triton X-100; pH 8; all components (Sigma-Aldrich, St. Louis, MO, USA) containing 20 mg/mL were added directly before the use of lysozyme (50 mg/mL) (Serva, Heidelberg, Germany). After the incubation of the mixture for 1 h at a temperature of 37 °C, 1 µL proteinase K (10 mg/mL, Sigma) was added and incubated for 1 h at a temperature of 56 °C. The lysates were boiled for 10 min to inactivate proteinase K and stored at a temperature of 4 °C.

With the thermal shift assay, thermal denaturation of the investigated protein was accessed as previously described [48 (link)]. The fluorogenic dye Sypro orange was mixed with the protein and heated stepwise. When the protein is folded correctly, the dye does not bind efficiently on the hydrophilic surface. When hydrophobic stretches are exposed due to denaturation of the protein, binding of the dye leads to an increase in fluorescence. For the assay, 10 µl protein samples (> 0.1 mg/ml) are mixed with 5 µl 50x SYPRO Orange (Sigma Aldrich) and 20 µl 10 mM HEPES pH 8.0. As a positive control, 10 mg/ml lysozyme (Serva) was used. Measurement was done with qTower3G and qPCRsoft 4.0 (Analytik Jena) using the TAMRA Channel (λ_ex = 535 nm, λ_em = 580 nm). The heating program was 25 to 95 °C with steps of 2 °C, 120 s hold time per temperature and a heating speed of 4.4 °C/s.

Proteins were overexpressed in BL21 competent E. coli for 4 h at RT after induction with 1 mM IPTG. Cells were lysed in 300 mM NaCl, 50 mM Tris/HCl pH 7.5, 5 mM EDTA, 5 mM EGTA, 0.01% (v/v) Igepal, protease inhibitors (cOmplete, EDTA-free protease inhibitor cocktail tablets, Roche), 50 mg/ml Lysozyme (Serva) and by sonication. After centrifugation at 10 000 g for 30 min the lysate was incubated with glutathione sepharose 4B (GE Healthcare) for 1.5 h at 4°C and subsequently washed 3 times with lysis buffer.

Plasmid DNA was isolated according to the protocol of Anderson and McKay [34 (link)] with the following modifications: after the addition of lysozyme (10 mg/mL, SERVA Electrophoresis GmbH, Heidelberg, Germany), 20 µL RNase A (100 mg/mL, Qiagen GmbH, Hilden, Germany) was added to the reaction mixes, which were incubated at 37 °C for 60 min. Phenol, which was saturated with TE-buffer and chloroform-isoamyl alcohol (both Carl Roth GmbH & Co. KG, Karlsruhe, Germany), was used for the protocol. Purified plasmid DNA was dissolved in 30 µL of TE-buffer and analyzed by electrophoresis on 0.8% (w/v) agarose gels.

The, E.coli strain DH5α was used for transformations that were performed like described previously [49 (link),50 (link)]. A. nidulans transformations were performed as described elsewhere [51 (link)]. The protoplastation was initiated with 15 mL 30 mg/mL Vinoflow^® Max or Vinotaste^® Pro from Novozymes (Bagsvaerd, Denmark) and 15 mg/mL lysozyme from Serva (Heidelberg, Germany) dissolved in citrate buffer (150 mM KCl, 580 mM NaCl, 50 mM Na-citrate, pH 5.5) for 105 min at 30 °C. Strains were verified by Southern hybridization [52 (link)]. Transformations of S. cerevisiae strain EGY48 were performed with the LiAc/SS Carrier DNA/PEG method [53 (link),54 (link)]. Plasmids used for the transformations are listed in Table S1, and resulting strains used or prepared in this study are listed in Table S2. All oligonucleotides used for the construction of plasmids are given in Table S3, and oligonucleotides used for qRT experiments are given in Table S4.

Amino acids, cultivation media components, Tris (tris(hydroxymethyl)aminomethane), metal salts and solvents were from Carl Roth (Germany), acetyl amino acids were obtained from Sigma-Aldrich. Reagents for molecular biology were from Thermo Fisher Scientific (USA). DNA oligonucleotide synthesis and DNA sequencing were performed by Eurofins Genomics (Germany). Strep-Tactin columns were from IBA (Germany). Products for isoelectric focusing and native PAGE, as well as lysozyme were purchased from SERVA (Germany). Other chemicals were purchased from Sigma-Aldrich (USA). The EZ Nin reagent for amino acid quantification was from Biochrom (UK).

Protein dye staining was performed as described5 (link). Briefly, phosphorylase b, bovine serum albumin (BSA), alcohol dehydrogenase (ADH; all three from Sigma), chymotrypsin and lysozyme (both from Serva) were dissolved at 10 mg/ml in 0.15 M sodium chloride. Remazol Brilliant Blue R (RBBR; Sigma), Remazol Brilliant Orange 3R (aka Reactive Orange 16; Sigma), Remazol Golden Yellow RNL, Remazol Brilliant Red F3B, Reactive Black 5 and Remazol Brilliant Violet 5R (all a kind gift of DyStar Textilfarben GmbH, Germany) were dissolved at 10 mg/ml in 10% w/v SDS. 200 μl of each protein solution was mixed with 50 μl of the respective dye solution and 50 μl of 1 M disodium hydrogen phosphate (Na₂HPO₄) pH 9.6 and incubated for 20 min at 65 °C. The Remazol dye stained proteins were separated by preparative SDS-PAGE, the bands corresponding to the stained proteins were excised from the gel and the proteins were electro-eluted using a Schleicher&Schuell Elutrap. Electro-eluted proteins were dialyzed for 14 hours at 4 °C against Tris-buffered saline (TBS).