Markerless gene deletion in Porphyromonas gingivalis using a pheS*-based counterselection system

Porphyromonas gingivalis is an oral pathobiont implicated in periodontitis and several systemic diseases and serves as an important model organism. However, the routine generation of markerless mutants in P. gingivalis has remained challenging due to the lack of an efficient counterselection system for the double cross-over approach. Markerless gene deletion is crucial for bacterial genetic manipulations, in particular for generating multiple gene deletions or introducing point mutations. In this study, a counterselection system for P. gingivalis was established by placing the pheS* gene under the control of a P. gingivalis promoter enabling efficient expression. The construct was delivered to P. gingivalis via a suicide plasmid by conjugation. Using PG0719 as a representative target gene, first cross-over recombinants were selected using erythromycin resistance encoded on the suicide plasmid. Cells were then subjected to counterselection in the presence of p-chloro-phenylalanine (p-Cl-Phe). Retention of pheS* in the genome reduced viability, thereby enriching recombinants that had undergone a second recombination event and loss of the plasmid sequence. Candidate clones were screened by colony PCR analysis to confirm the loss of the gene of interest. A markerless PG0719 mutant was generated and further validated by Sanger sequencing, demonstrating a practical approach for markerless gene deletion in P. gingivalis and providing a framework for further genome modifications in the organism.IMPORTANCEAlthough Porphyromonas gingivalis is a widely studied model organism, the genetic manipulation of this bacterium has remained limited by the lack of efficient tools for markerless genome editing. Here, we established a counterselection system based on pheS^∗ that enables markerless gene deletion in P. gingivalis. This approach addresses a technical limitation in the field and provides a practical and broadly applicable framework for advanced genetic manipulation in this important oral pathobiont.