Aug 2026· Microbiology spectrum· Vol 14· 0 citations· 35 references
Medicine
TL;DR
A counterselection system based on pheS∗ that enables markerless gene deletion in P. gingivalis is established and provides a practical and broadly applicable framework for advanced genetic manipulation in this important oral pathobiont.
Abstract
ABSTRACT 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. IMPORTANCE Although 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. Although 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.
This chapter provides a detailed, step-by-step protocol for implementing a conditional plasmid system that enables efficient, markerless gene deletion in FNA strains and provides a powerful and adaptable tool for advancing genetic studies in this genetically recalcitrant subspecies.
B. G. C., Chenggang Wu· Methods in molecular biology· 0 citations
A second-generation genetic toolbox based on leucine auxotrophy that enables antibiotic-free positive selection in Mycobacterium abscessus and significantly reduces dependency on antibiotics for genetic manipulation, aligning with the goals of sustainable research and offering new opportunities for studying this clinically significant pathogen.
Tizian Griesser, P. Selchow, Peter Sander· Applied and Environmental Mi...· 0 citations
Abstract
Background Porphyromonas gingivalis (P. gingivalis) is a Gram-negative anaerobic bacterium and a keystone pathogen in chronic periodontitis. Beyond oral disease, it has been implicated in systemic conditions, including cardiovascular disease, type 2 diabetes, rheumatoid arthritis, and neurodegeneration. Marked strain-to-strain differences in tissue invasion, immune evasion, and pathogenicity suggest that virulence is shaped by complex genetic and regulatory interactions rather than a single determinant. Objective To summarize current knowledge of the genetic diversity of P. gingivalis virulence determinants and their contribution to strain-specific pathogenicity. Design This narrative review synthesizes current evidence on the diversity of major virulence determinants, including fimbriae (FimA and Mfa1), gingipains (RgpA/RgpB/Kgp), Hag-family hemagglutinins, capsular polysaccharide loci, lipopolysaccharide variants, the RagAB nutrient acquisition system, and the type IX secretion system. It also examines the roles of allelic variation, domain rearrangements, phase variation, and horizontal gene transfer in shaping strain-specific virulence repertoires. Conclusions The reviewed evidence indicates that genetic variation across multiple virulence-associated loci contributes to substantial phenotypic diversity among P. gingivalis strains, influencing host interaction, tissue tropism, immune modulation, and pathogenic potential. Integrating comparative genomics with functional phenotyping provides a framework for predicting strain-specific virulence and may facilitate the development of improved diagnostic tools and targeted therapeutic strategies.
Hanie Ahmadi, J. Burks, Katherine A. Innamorati et al.· Journal of Oral Microbiology· 1 citation
ABSTRACT The global spread of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a critical clinical threat, with blaKPC as a key mobile carbapenem resistance gene. Here, we investigated a novel blaKPC-2 genetic element and its transmission in clinical ST2483 isolate PA100 via whole-genome sequencing, molecular, and phenotypic testing, and vesicle analysis. A unique 4,746-bp IS26-flanked structure inserted at the chromosomal InaA/pncB2 locus was confirmed, which forms a circular transposition intermediate through IS26 recombination. PA100 exhibited ~40-fold higher blaKPC-2 copy number and ~25-fold higher mRNA expression vs chromosomal blaKPC-2 strain PA3; meropenem exposure further elevated its transcription by nearly 70-fold, far exceeding controls. PA100 showed an extensively drug-resistant phenotype with high-level carbapenem resistance, while remaining susceptible to ceftazidime-avibactam and cefiderocol. No classical conjugation or transformation was observed, while intact outer membrane vesicles (OMVs) from PA100 carried blaKPC-2, supporting a non-plasmid, vesicle-associated route for transient delivery of the resistance gene under the tested conditions. The circular transposon remained genetically stable after 10 generations of passaging in PA100; however, stable maintenance in the PAO1 recipient was not detected after passage. In summary, we first report an IS26-driven circular blaKPC-2 transposon in ST2483 P. aeruginosa, conferring robust overexpression and stable inheritance in the donor strain, with OMVs potentially facilitating its horizontal spread, although stable maintenance in recipient cells was not demonstrated under the current assay conditions. These data advance understanding of blaKPC dissemination in CRPA and highlight clinical surveillance priorities for rare high-risk sequence types.
Mingxiao Chen, Qingqing Zhi, Miaoshan Luo et al.· Antimicrobial Agents and Che...· 0 citations
Nontuberculous mycobacteria (NTM) are emerging pathogens for which genetic tools remain limited. Here, we developed an arabinose-inducible gene expression system based on a modified pBAD24 vector adapted for mycobacterial hosts. The vector carries replication origins for mycobacteria and Escherichia coli, as well as selectable markers compatible with NTM. In Mycobacterium abscessus (Mycobacteroides abscessus), the system enabled dose-dependent induction of target gene expression by arabinose, as demonstrated by increased antibiotic resistance and quantitative RT-PCR analysis. Although basal expression was observed in the absence of arabinose, expression levels were tunable across arabinose concentrations. The system was also functional in Mycobacterium smegmatis (Mycolicibacterium smegmatis) and Mycobacterium bovis BCG, although the degree of basal expression varied among host species. These results establish a tunable inducible expression system for mycobacteria and provide a useful genetic tool for studies of NTM biology.
Yuya Yanagita, Mai Maruhashi, Kotaro Sawai et al.· Journal of Microbiological M...· 0 citations
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