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. 2024 Oct 21:15:1476171.
doi: 10.3389/fmicb.2024.1476171. eCollection 2024.

A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence

Kimihiro Abe  1   2 , Hiroko Yahara  3 , Ryoma Nakao  1 , Takehiro Yamaguchi  1 , Yukihiro Akeda  1
Affiliations

A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence

Kimihiro Abe et al. Front Microbiol. .

Abstract

Porphyromonas gingivalis is a major oral bacterial pathogen responsible for severe periodontal diseases. Numerous studies have used genetic approaches to elucidate the molecular mechanisms underlying its pathogenicity. Typically, electroporation and conjugation are utilized for mutagenesis of P. gingivalis; however, these techniques require specialized equipment such as high-voltage electroporators, conjugative plasmids and donor strains. In this study, we present a simple, cost-effective transformation method for P. gingivalis without any special equipment by exploiting its natural DNA competence. P. gingivalis ATCC 33277 was grown to the early-exponential phase and mixed with a donor DNA cassette. This mixture was then spotted onto a BHI-HM blood-agar plate and incubated for one day to promote colony biofilm formation. The resulting colony biofilm was suspended in a liquid medium and spread onto antibiotic-containing agar plates. Transformants appeared within 4 to 5 days, achieving a maximum efficiency of 7.7 × 106 CFU/μg. Although we optimized the transformation conditions using a representative strain ATCC 33277, but the method was also effective for other P. gingivalis strains, W83 and TDC60. Additionally, we discovered that deletion of PGN_0421 or PGN_0519, encoding putative ComEA and ComEC, abolished competency, indicating that these gene products are essential for the natural competence.

Keywords: Porphyromonas gingivalis; genetic engineering; horizontal gene transfer; natural competence; transformation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic of the transformation procedure. (i) Culture P. gingivalis at 37°C in 5 mL of BHI-HM under anaerobic conditions until the cell growth reaches the early-mid exponential phase (at OD600 of approximately 0.3; 10–16 h of cultivation). (ii) Harvest the cells by brief centrifugation and resuspend the pellet in 500 μL of fresh, prewarmed BHI-HM. (iii) Mix 20 μL of the cell resuspension with 100 ng of donor DNA containing 1,000-bp homology arms, spot the mixture on a BHI-HM blood-agar plate, and incubate at 37°C for 24 h under anaerobic conditions. (iv) Collect and suspend the colony biofilm in 500 μL of fresh, prewarmed BHI-HM and spread the suspension on an antibiotic-containing BHI-HM blood-agar plate. (v) Incubate the plate at 37°C under anaerobic conditions for 4–5 days. These are the optimized conditions for transformation determined in this study.
Figure 2
Figure 2
Natural competence-mediated integration of ermF into P. gingivalis genome. (A) Schematic representation of the integration of an erythromycin-resistant gene (ermF)-harboring donor DNA into the P. gingivalis ATCC 33277 genome via double homologous recombination. The ermF donor DNA includes 500-bp homology arms for integration into the intergenic region between PGN_0032 and PGN_0033. Arrows indicate the positions and orientations of PCR primers used for genotyping. (B) Colony PCR confirmation. The integration of ermF in the transformant genome was confirmed by colony PCR, examining nine colonies with the primers shown in (A). A wild-type colony served as a negative control.
Figure 3
Figure 3
Effects of culture duration on transformation efficiency. (A) Cell growth. An optical density at 600 nm of P. gingivalis ATCC 33277 cultured at 37°C in liquid BHI-HM was measured at 6, 10, 16, 24, 34, and 42 h after inoculation. (B) Impact of cell growth phase on transformation efficiency. Cells harvested at the time points indicated by red arrows in (A) were transformed with 100 ng of ermF donor DNA with 1,000-bp homology arms. The colony biofilm was developed for 24 h. Transformation efficiency was assessed as the number of erythromycin-resistant (Emr) colonies per ml of the biofilm suspension (CFU/ml). Error bars represent ±standard deviations from three independent experiments. (C) Effect of biofilm culture duration on transformation efficiency. P. gingivalis recipient cells in the early-mid exponential phase were mixed with 100 ng of donor DNA with 1,000-bp homology arms and spotted on agar plates. Colony biofilms collected at the specified time points were spread on BHI-HM blood-agar plates containing erythromycin. Transformation efficiency was quantified as CFU/ml of Emr colonies. Error bars represent ±standard deviations from three independent experiments.
Figure 4
Figure 4
Influence of donor DNA amounts and homology arm length on transformation efficiency. (A) Donor DNA amounts. P. gingivalis recipient cells in the early-mid exponential phase were transformed with varying amounts of donor DNA containing 1,000-bp homology arms. Colony biofilms were developed for 24 h. Transformation efficiency was measured as CFU/ml of Emr colonies. Error bars represent ±standard deviations from three independent experiments. ND, Not Detected. (B) Homology arm length. P. gingivalis recipient cells in the early-mid exponential phase were transformed with 100 ng of donor DNA containing various homology arm lengths. The colony biofilm was developed for 24 h. Transformation efficiency was quantified as CFU/ml of Emr colonies. Error bars represent ±standard deviations from three independent experiments.
Figure 5
Figure 5
Gene deletions of comEA and comEC in P. gingivalis ATCC 33277. (A) Schematic of gene replacements for comEA (PGN_0421) and comEC (PGN_0519) with cepA via recombination. The cepA cassette contains 0.5-kb homology arms at the 5′ and 3′ ends corresponding to the upstream and downstream regions of comEA (or comEC). Arrows indicate the positions of PCR primers with the orientations used for genotyping. (B) PCR confirmation. The comEA and comEC deletion mutants were verified by PCR using combinations of cepA-specific and genome-specific primers: Up, upstream region-specific forward primer for comEA (or comEC) combined with cepA-specific reverse primer; Down, cepA-specific forward primer combined with downstream region-specific reverse primer for comEA (or comEC). WT, P. gingivalis ATCC 33277; ΔEA, ΔcomEA; ΔEC, ΔcomEC. (C) Transformation efficiency of comEA and comEC mutant strains. Transformation assays were conducted using 100 ng of ermF donor DNA with 1,000-bp homology arms under the same conditions as in Figure 1. Transformation efficiency was quantified as CFU/ml of Emr colonies. Error bars represent ±standard deviations from three independent experiments. ND, Not Detected.
Figure 6
Figure 6
Transformation efficiency of P. gingivalis strains W83 and TDC60. Transformation assays of P. gingivalis ATCC 33277, W83, and TDC60 were conducted using 100 ng of ermF donor DNA with 1,000-bp homology arms under the conditions described in Figure 1. Transformation efficiency is shown as CFU/ml of Emr colonies. Error bars represent ±standard deviations from three independent experiments.
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by MEXT KAKENHI Grant Number 20H03861 (to RN) and by JSPS KAKENHI Grant Numbers 21K18284, 21KK0164 (to RN).

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