Random Mutagenesis in <i>Porphyromonas gingivalis</i> Enables Isolation of Mutants With Enhanced Secreted Protease Activity

Porphyromonas gingivalis is a key pathogen in periodontitis, with secreted proteases as major virulence factors. We developed a screening method to generate and identify P. gingivalis mutants with elevated protease activity. Mutations were induced using the mutagens 2,6-diaminopurine (2,6-DAP) or ethyl methanesulfonate (EMS), and the mutagenized cells were subsequently plated on casein agar. During colony growth, the medium became opaque due to partial casein precipitation, whereas colonies with higher protease activity produced clear halos through casein degradation. Colonies that formed halos earlier than the wild type were selected for further analysis. Liquid culture assays of the supernatants identified four strains with enhanced protease activity, of which two were 2,6-DAP-derived and two were EMS-derived. Whole-genome sequencing revealed that the two 2,6-DAP-derived strains carried mutations in iron transport-related genes (foeA and tonB, respectively), likely increasing protease levels through iron limitation-induced upregulation of rgpA. The two EMS-derived strains contained multiple mutations, including one in rgpA, a major protease gene. The N-terminal region of RgpA, which contains the protease motif, harbored the G450D mutation in one strain and the C600Y mutation in the other. These results demonstrate that our method efficiently generates P. gingivalis mutants with protease gene alterations that increase enzymatic activity. This approach provides a useful tool for studying protease function and virulence mechanisms in this pathogen, and for identifying genes that affect protease secretion.