Secretome Analysis

After cultures of B. subtilis natto reached stationary phase, cultures were centrifuged (5000 × g, 30 min) and cell-free supernatant (CFS) was carefully removed by pipetting into a fresh tube. Control secretome experiments used the CFS from BFC experiments, prepared in the same way. After collecting the CFS, aliquots were washed twice in H₂O using Amicon Ultra 0.5 Centrifugal Filter Units with 10 kDa nominal molecular weight cut-off (Merck, Darmstadt, Germany). A Bradford Protein Assay was performed to measure the protein concentration. Briefly, Bradford Dye Reagent (Bio-Rad Laboratories, CA, United States) was diluted 1:4 in H₂O. In a 96-well plate, 10 μL of CFS were mixed with 200 μL of diluted Bradford Dye Reagent and incubated at room temperature for 5 min. Absorbance was measured at 595 nm using a BMG Labtech CLARIOstar spectrophotometer (BMG labtech, CA, United States). Protein concentration was quantified by comparison to a standard curve produced using Bovine Serum Albumin (Bio-Rad Laboratories, CA, United States).

To visualize the proteins present, CFS samples were analyzed via SDS-PAGE. For this, 5 mL of CFS was concentrated to ∼500 μL using Amicon Ultra 0.5 Centrifugal Filter Units with 10 kDa nominal molecular weight cut-off (Merck, Darmstadt, Germany). Approximately equal total amounts of protein were loaded onto an SDS-PAGE gel, dyed using InstantBlue Stain (Sigma-Aldrich, MO, United States).

To identify proteins visualized by SDS-PAGE, trypsin in-gel digestion was performed as described previously in Srivastava et al. (2013). Briefly, bands of interest were cut out of the gel and diced into ∼1 mm³ pieces. These were incubated in 200 μL gel reagent (50% 0.1 M ammonium carbonate, 50% acetonitrile, pH 10) at 37°C for 60 min. Gel pieces were dehydrated by mixing with 100% acetonitrile. Next, 100 μL of a 10 mM solution of DTT in 50 mM ammonium bicarbonate was added to the gel pieces, and incubated for 30 min at 60°C. After removing and discarding excess supernatant, 30 μL of 1% iodoethanol solution in 50 mM ammonium bicarbonate was added for a 15-minute incubation at 37°C. Dehydration with 100% acetonitrile was repeated. To digest proteins in the gel, 30 μL trypsin solution (comprising trypsin (10 ng/μL) and Protease Max (0.01%) in 50 mM ammonium bicarbonate) was added to dehydrated pieces, for incubation on ice for 15 min. Excess trypsin solution was removed, and 50 μL of 50 mM ammonium bicarbonate solution was added for overnight incubation at 37°C. Supernatant containing released peptides was transferred to a fresh tube. Additional peptides were extracted from the gel pieces using 50 μL formic acid solution (5% formic acid, 50% acetonitrile, 45% H₂O), and incubated for 5 min at room temperature. Peptides were speed-vacced to dryness and stored at −20°C until analysis by mass spectrometry.

Peptide analysis and identification was performed as described by Srivastava et al. (2013). A reversed-phase liquid chromatography electrospray ionization mass spectrometer (LC-ESI-MS/MS), using a nanoACQUITY ultra-performance liquid chromatography (UPLC) system coupled to a Q-TOF mass spectrometer (Xevo Q-TOF; Waters, Milford, MA, United States) was used. Briefly, peptides were loaded onto a C18 trap column (Symmetry 180 μm × 20 mm, 5 μm; Waters, Milford, MA, United States) followed by washing with 1% (v/v) acetonitrile and 0.1% (v/v) formic acid at 10 μL min^–1 for 5 min. The samples eluted from the trap column were separated on a C18 analytical column (75 μm × 100 mm, 1.7 μm; Waters, Milford, MA, United States) at 250 nl min^–1 using 0.1% formic acid as solvent A and 0.1% formic acid in acetonitrile as solvent B in a stepwise gradient: 0.1%–10% B (0–5 min), 10–30% B (5–32 min), 30–40% B (32–35 min), 40–85% B (36–38 min), 85% B (38–40 min), 85–0.1% B (40–42 min), and 0.1% B (42–60 min). The eluting peptides were sprayed in the mass spectrometer (capillary and cone voltages set to 2.1 kV and 35 V, respectively), and MS/MS spectra were acquired using automated data-directed switching between the MS and MS/MS modes using the instrument software (MassLynx V4.0 SP4). The three most abundant signals of a survey scan (400–1600 m/z range, 1 s scan time) were selected by charge state, and collision energy was applied accordingly for sequential MS/MS fragmentation scanning (50–1800 m/z range, 1 s scan time). The MS raw data files were processed using Mascot Distiller (version 2.4.3.2, Matrix Science, London, United Kingdom) and the resulting files were submitted to a local Mascot (Matrix Science, version 2.3.1) server using the NCBI database with both B. subtilis (1084562 sequences) and general fungi (5915770 sequences) taxonomies. The following settings were used for the database search: trypsin-specific digestion with one missed cleavage allowed, ethanolylated cysteine as fixed and oxidized methionine as variable modifications, peptide tolerance of 100 ppm, and fragment tolerance of 0.6 Da. Peptides with Mascot ion scores exceeding the threshold for statistical significance (p < 0.05) were approved. Only proteins identified by two or more unique peptides were selected.