Stability Analysis of S1 Protein in Mutant Strain

The mutant strain S1/ΔAlr HLJ-27 was cultured overnight in MRS medium supplemented with 200 μg/mL d-alanine, and the bacterial precipitate was collected by centrifugation at 10,000 g for 2 min. The precipitate was incubated with 1% lysozyme and sonicated for 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) detection and western blotting. SDS-PAGE was used to separate the proteins in the mixture, which were then transferred onto PVDF membranes (Millipore, Milford, MA, USA) using a mouse anti-S1 monoclonal antibody (mAb cell supernatant) prepared in our laboratory as the primary antibody (Xiao et al. 2022 (link)). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Sigma, Ronkonkoma, NY, USA) was used as the secondary antibody at a dilution of 1:5000. The results were evaluated using a chemiluminescent substrate reagent (Thermo Scientific, Durham, NC, USA) according to the manufacturer’s instructions. To determine the stability of S1 gene expression in the mutant strain S1/ΔAlr HLJ-27, protein samples for the western blot assay were obtained with every five generations of the strain within 20 generations. The strain ΔAlr HLJ-27 was used as a negative control.