Assessing the Antimicrobial Activity of Nisin Variants Using an Agar Well Diffusion Assay

Abstract

Source: Nickling, J. H., et.al. Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids. J. Vis. Exp. (2018)

This video demonstrates an agar well diffusion assay used to evaluate the antimicrobial activity of nisin variants. It shows how Lactococcus lactis expressing the membrane-bound peptidase NisP activates nisin precursors produced in E. coli, resulting in growth inhibition halos. The size of these halos reflects the bioactivity of each nisin variant.

Protocol

1. Antimicrobial Activity Test

1. Preparation of GM17-agar plates under sterile conditions
   1. Prepare an overnight culture of the indicator strain L. lactis NZ9000 carrying plasmid pNG nisPT at 30 °C in M17 broth with 1% (w/v) glucose (=GM17) and 5 µg/mL chloramphenicol.
   2. Measure OD₆₀₀, inoculate fresh medium to OD₆₀₀ = 0.1 and incubate until OD₆₀₀ = 0.4-0.6. Then place the flask on ice.
      NOTE: Each OD₆₀₀ measurement will consume culture volume. Keep in mind that for each assay agar plate, 1 mL bacterial culture will be needed. If required, scale up the liquid culture volume accordingly.
   3. For 1.5% agar, weigh out 4.5 g agar in a glass media bottle. Add 300 mL ddH₂O, mix, and autoclave.
   4. Prepare 2x M17 broth (two-times concentrated) in 300 mL ddH₂O and autoclave.
   5. Mix 25 mL 2x M17 broth containing 10 µg/mL chloramphenicol and 2% glucose with 1 mL L. lactis preculture (4% v/v).
   6. Add 25 mL molten 1.5% agar (freshly autoclaved or heated in a microwave).
      NOTE: Prior to this, let the bottle cool to the touch (around 50 °C). This is necessary since L. lactis is a mesophilic organism sensitive to high temperatures.
   7. Pour the solution into a large petri dish. Dry plates for 10-15 min.
   8. Sterilize the ends of a glass Pasteur pipette by flame. Wait for it to cool down, then use the wide end to create holes in the solidified GM17-agar.
2. Sample preparation
   1. Take 1 mL of the E. coli expression cultures in a labelled 1.5 mL tube and centrifuge for 3 min at 7,000 x g. Aspirate the remaining medium and resuspend the cell pellet in 500 µL Na-P (50 mM sodium phosphate buffer, pH 7.4 made from 0.5 M sodium dihydrogen phosphate and 0.5 M disodium hydrogen phosphate).
   2. Sonicate the samples on ice. Submerge the tip of the sonicator probe into the cell suspension. Set sonicator at 30 % amplitude with pulse of 1 s on and 5 s off for 3 min.
   3. Centrifuge the cell lysate for 10 min at 13,000 x g to pellet cell debris. Transfer the supernatant to a new reaction tube on ice.
   4. Dilute and normalize the cell extract supernatants to 1 mL OD₆₀₀ = 0.6, relative to the harvested cell density, with Na-P.
3. Activity test
   1. Add 40 µL of each normalized sample into a hole of the indicator agar plate (Figure 1). Use chloramphenicol at 400 µg/mL as antibacterial control compound. Use elution buffer as a negative control. Wait until all samples are diffused into the agar. Incubate the plate overnight at 30 °C.
   2. Take pictures of the agar plates using a flatbed scanner or digital camera. Growth inhibition halo sizes can be measured by hand or using ImageJ.

Results

Figure 1. Antimicrobial activity assay of cell lysates containing nisin variants produced

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium dihydrogen phosphate monohydrate	Carl Roth, Germany	K300.2
Disodium hydrogen phosphate	Carl Roth, Germany	P030.2
D-glucose	Carl Roth, Germany	6780	Prepare a 20% (w/v) solution for addition into molten agar
Chloramphenicol	Carl Roth, Germany	3886.1	Working concentration 5 µg/mL for L. lactis, prepare 37 mg/mL stock in ethanol
M17-broth	Sigmal-Aldrich, Germany	56156	Commercial product
Agar-agar	Carl Roth, Germany	5210.5
Nisin from Lactococcus lactis	Sigma-Aldrich, Germany	N5764	Commercial product, can be used as reference for bioactivity
1.5 mL Eppendorf tubes	Eppendorf, Germany	30120086
Petri dishes (polystyrene, sterile)	Carl Roth, Germany	TA19
Lactococcus lactis NZ9000 pNG nisPT			Bacterial indicator strain
Benchtop centrifuge for 1.5 mL Eppendorf tubes	Eppendorf, Germany	5427 R
Probe sonicator	Bandelin, Germany	Sonopuls HD3200 with sonotrode MS-72	200 W maximum HF output