Investigating the Bacterial Response to Ethylene Using an Ethylene-Releasing Compound

Samenvatting

Source: Augimeri, R. V. et. al., Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria. J. Vis. Exp. (2016)

This video demonstrates the effect of ethylene generated by 2-chloroethylphosphonic acid (CEPA) on bacterial growth and cellulose production. Ethylene activates signaling pathways that stimulate cellulose synthesis, resulting in enhanced pellicle formation. These results highlight the bacterial response to ethylene.

Protocol

1. Pellicle Assays
   1. Grow and Quantify Komagataeibacter xylinus Starter Cultures in Triplicate:
      1. In triplicate, inoculate a single colony of Komagataeibacter xylinus into 5 ml of Schramm and Hestrin (SH) medium (pH 5) supplemented with 0.2% (v/v) filter-sterilized cellulase. Incubate cultures at 30 °C with agitation at 150 rpm until an optical density at 600 nm (OD₆₀₀) of 0.3 to 0.4 is reached (about 72 hr).
      2. Harvest starter cultures by centrifugation (2,000 x g; 4 °C; 10 min). Wash the cells twice with 5 ml of sterile saline and resuspend the cell pellet. Keep the cells on ice.
      3. Quantify cells using a Petroff-Hausser counting chamber.
   2. Prepare Master Mixes to Inoculate 24-well Plates:
      1. Supplement 60 ml of SH medium (pH 7) with 120 µl of the 5, 50, and 500 mM 2-chloroethylphosphonic acid (CEPA) stocks to obtain final CEPA concentrations of 0.01, 0.1, and 1.0 mM, respectively. Supplement another 60 ml of SH medium (pH 7) with 120 µl of the solvent used to dissolve CEPA. Vortex to mix.
      2. Divide each 60 ml of CEPA-containing medium into four 14 ml aliquots. Inoculate three of the 14 ml aliquots with biological replicates of the starter culture at a concentration of 10⁵ cells/ml. Keep tubes with cells on ice to prevent cellulose production.
         NOTE: The remaining 14 ml aliquot will be used for sterile control wells.
   3. Inoculate 24-well Plates:
      1. Test each treatment in its own plate with three rows of biological replicates and a row of sterile controls (Figure 1).
      2. Using the 14 ml master mix, add 2 ml into each of six wells of a sterile 24-well plate. Complete for the three biological replicates and sterile control (Figure 1). Repeat for each treatment.
      3. Seal plates with paraffin film and incubate statically for 7 days at 30 °C.
      4. Harvest and measure pellicle dry Weight (Cellulose Yield) (Figure 1):
         1. Individually transfer the pellicles into the wells of a 6-well plate. To lyse cells, treat the pellicles with 12 ml of 0.1 N sodium hydroxide (NaOH) at 80 °C for 20 min.
         2. Remove the NaOH and neutralize pellicles by washing with ultra-pure water for 24 hr with agitation. Change water every 6 hr.
            NOTE: Pellicles should be white upon completion of the washing step.
         3. Place pellicle weights on silicon mats and dry at 50 °C for 48 hr to constant weight. Once dry, remove them from the mats and measure their weights on an analytical scale to determine bacterial cellulose yield.

Resultaten

Figure 1: Flow chart illustrating the protocol used for pellicle assay and analysis. Stock CEPA-supplemented pH 7 SH medium ...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Cellulase from Trichoderma reesei ATCC 26921	Sigma	C2730	Aqueous solution
Citric acid	BioShop	CIT002.500	For SH medium
Ethephon (≥ 96%; 2-chloroethylphosphonic acid)	Sigma	C0143	Ethylene-releasing compound
Glucose	BioBasic	GB0219	For SH medium
Komagataeibacter xylinus ATCC 53582	ATCC	53582	Bacterial cellulose-producing alphaproteobacterium
Sodium phosphate, dibasic heptahydrate	BioShop	SPD579.500	Sodium phosphate, dibasic heptahydrate for SH medium
NaCl	BioBasic	SOD001.1	Sodium chloride for saline and control solution
Sodium phosphate, monobasic monohydrate	BioShop	SPM306.500	Sodium phosphate, monobasic monohydrate for control solution
NaOH	BioShop	SHY700.500	Sodium hydroxide for pH adjustment
Paraffin film	Parafilm	PM996	For sealing plates and flasks
Peptone (bacteriological)	BioShop	PEP403.1	For SH medium
Petroff-Hausser counting chamber	Hausser scientific	3900	Bacterial cell counting chamber
Polyethersulfone sterilization filter 0.2 µm	VWR	28145-501	For sterilizing cellulase
Yeast extract	BioBasic	G0961	For SH medium