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1. Natural Transformation
NOTE: The N315 derivative, N2-2.1 was used as the recipient. In this strain, the sigH locus was duplicated so as to constitutively express SigH. If the resistance marker to be transferred is not chloramphenicol, pRIT-sigH (CmR) can be used to express SigH. Purified plasmid or whole DNA extract from cfr-acquired S. aureus COL-45 is used as the donor DNA for transformation.
- Preparation of donor DNA
- Cultivate COL-45 overnight with shaking at 37 °C in a 300 ml flask containing 50 mL of Tryptic soy broth (TSB) supplemented with 32 mg/L chloramphenicol. Culture E. coli HST04 carrying pHY300 plasmid(TetR) to extract the plasmid for the control experiment.
NOTE: HST04 (dam-/dcm-) lacks the DNA methylase genes. Other conventional strains with DNA methylases can also be used to prepare the DNA donor, because the DNA methylation state does not affect the transformation efficiency. Alternatively, pT181 plasmid purified from the S. aureus COL strain can also be used as a positive control for the transformation assay. - Collect the cells by centrifugation (8,000 x g for 10 min at 4 °C).
- Extract the plasmids using a plasmid DNA extraction kit or a conventional DNA purification method to purify the whole DNA.
- Quantify the purified DNA by spectrometer and keep it at 4 °C until use.
NOTE: Use a fresh DNA preparation for the transformation assay, usually less than one week old. Old DNA preparations reduce the efficiency of transformation.
- Transformation assay
- Culture the recipient cell (N2-2.1) overnight in 5 ml of TSB at 37 °C with shaking.
- Transfer 0.5 ml of the overnight culture into a 1.5 ml tube. Precipitate the cells by centrifugation (10,000 x g for 1 min at 4 °C).
- Suspend the cells with 10 mL of CS2 medium in a 50 ml tube.
NOTE: CS2 medium is a complete synthetic medium that induces competence for natural transformation in S. aureus. Other standard laboratory media, such as TSB or Brain heart infusion (BHI), are not suitable for transformation. - Grow the bacteria at 37 °C with shaking (180 rpm) until the late exponential phase (about 8 hr).
- Harvest the cells by centrifugation (5,000 x g for 5 min at 4 °C).
- Resuspend the cells in 10 ml of fresh CS2 medium.
- Add 10 µg of purified plasmid or genome DNA (step 1.1.4) to the cell suspension. Shake at 37 °C and 180 rpm for 2.5 hr.
NOTE: Shorter incubation times with DNA (<2.5 hr) result in lower transformation frequencies. - Collect cells by centrifugation (5,000 x g for 5 min at 4 °C).
- Resuspend the cells in 10 mL of BHI medium. Mix the cell suspension with 90 ml of melted BHI agar (55 °C) supplement with 32 mg/L chloramphenicol (or 5 mg/L tetracycline in the control experiment). Pour the mixture into the 90 mm Petri dishes. Swiftly cool and let the agar solidify.
- Incubate the plates at 37 °C for 2 days.
- Replicate generated colonies (transformants) by transferring the colonies (using toothpicks) to new BHI agar plates containing the appropriate antibiotics to confirm their resistance characteristics. Confirm the acquired resistance gene (cfr or tetM) by colony PCR.