Pediococcus damnosus is a lactic acid bacterium often found in wine and beer. It produces the antimicrobial peptide pediocin PD-1, which is active against several lactic acid bacteria(1)
. P. damnosus contains several plasmids, some of which are more than 50kb and of various copy numbers. Our goal was to isolate one of the 50kb plasmids.
Several factors complicate plasmid DNA isolation from P. damnosus. Firstly, it is a Gram-positive bacterium with a different cell wall composition than E. coli, for which most plasmid extraction protocols were established. However, this can easily be overcome by performing a 30- to 60-minute pretreatment with lysozyme to degrade the thick peptidoglycan layer of the cell wall (2)
. Secondly, P. damnosus is grown in de Man, Rogosa and Sharpe (MRS) broth with Tween® 80 (Biolife, Italy, Cat.# 401729) at low pH rather than in Luria-Bertaini broth, which also could influence DNA isolation. Finally, P. damnosus contains several low-copy-number plasmids larger than 50kb, which have a tendency to change conformation or disappear completely, depending on the treatment.
Previous trials in our lab to isolate the plasmid using various miniprep protocols and kits failed to provide sufficient yield (Figure 1, Panel A). Attempts to pool and concentrate the samples via vacuum centrifugation (Centrivap Concentrator; Labconco, USA) led to a change in conformation of the large plasmids and retention of DNA in the wells of the agarose gel. Alternatively, DNA was lost during ethanol precipitation. When using column-based midiprep kits, the columns of the kit were clogged, causing the eluted DNA to run as a smear instead of distinct bands on the agarose gel, and much of the DNA remained in the wells (Figure 1, Panel B).
Adapting the PureYield™ Midiprep Protocol for P. damnosus
The purification protocol was established for P. damnosus DSM20331 using the Universal 32R centrifuge (Hettich, Germany) with a gmax of 3,857 and a swinging bucket rotor. Therefore, all centrifugation steps were performed at room temperature at 3,857 × g instead of 5,000 × g, as recommended. The DNA Purification by Vacuum protocol described in the PureYield™ Plasmid Midiprep System Technical Manual #TM253 (3)
, was used with modifications to the following steps:
Modified Step 1. Grow a 50ml culture of P. damnosus in MRS broth with Tween® 80 (pH 5.2 at 30°C; Biolife, Italy, Cat.# 401729). Grow for 2–3 days (at 30°C with shaking at 150rpm) to an O.D.600 of 1.5–2 (a higher O.D. leads to clogging of the PureYield™ Columns).
Modified Step 3. Resuspend cell pellets in 3ml of Cell Resuspension Solution. Add 50µl of lysozyme (200mg/ml, AppliChem, Germany), and incubate at 37°C for 30–60 minutes.
Modified Step 5. Add 5ml of Neutralization Solution to the lysed cells, cap the tube and mix by gently inverting the tube 3–5 times. Allow the lysate to sit for 2–3 minutes in an upright position to allow a white flocculent precipitate to form. It is important to wait for the precipitate to form to ensure thorough lysate clearing. Note: To ensure complete passage of the lysate through the PureYield™ Clearing Column and quick passage of the lysate and subsequent wash solutions through the PureYield™ Binding Column, centrifuge the lysate for 10 minutes before carefully applying the supernatant to the Clearing Column.
Modified Step 13. Remove the PureYield™ Binding Column from the vacuum manifold. Tap the tip of the column on a paper towel to remove excess ethanol. Wipe any excess ethanol from the outside of the tube.
Modified Step 14. Elute the plasmid DNA in 400µl of Nuclease-Free Water using the Eluator™ Vacuum Elution Device (Cat.# A1071) and following Steps 15–18 of the Elute by Vacuum protocol in the PureYield™ Plasmid Midiprep System Technical Manual #TM253 (3)
. The lower elution volume results in a more concentrated sample.
In a first attempt to isolate P. damnosus plasmid DNA, the DNA Purification by Quick Combination Method in the PureYield™ Plasmid Midiprep System Technical Manual #TM253 (3)
was followed using the Universal 32R centrifuge. Steps 1 and 3 were modified as described above to grow the P. damnosus cells at optimal conditions and perform a preincubation step with lysozyme. The lysate only partially passed through the Clearing Column, and the flowthrough remained turbid. Furthermore, elution by centrifugation was a complete failure. Only after adding 600µl of Nuclease-Free Water to the column, heating the column and the water together at 60°C for 5 minutes and centrifuging at maximum speed (3,857 × g) for 10 minutes could 80µl (approximately 5µg yield) of pure plasmid DNA, which did not smear on the agarose gel, be recovered.
Since it was clear that the centrifuge was not powerful enough, the DNA Purification by Vacuum protocol in the PureYield™ Plasmid Midiprep System Technical Manual #TM253 (3)
was performed using the Eluator™ Vacuum Elution Device for elution to increase plasmid DNA yield. Using the Eluator™ Vacuum Elution Device resulted in an almost complete recovery of the Nuclease-Free Water and thus a much higher yield of approximately 22µg at a concentration of approximately 60ng/µl. In addition, the plasmid bands can be distinguished clearly on the agarose gel, and only a small amount remained in the wells (Figure 2).
Further attempts to increase plasmid DNA yield and concentration by either increasing the culture volume to 100ml or by increasing the O.D.600 of the cells led to clogging of the PureYield™ Columns and a reduction in yield.
The yield and concentration of plasmid DNA obtained from P. damnosus using the PureYield™ Plasmid Midiprep System and the protocol described here may not match those from E. coli, but the isolated plasmid DNA is of high purity and sufficient for subsequent cloning experiments. Using this Gram-positive bacterium, we were able to isolate 22µg of DNA from a 50ml culture.