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

Abstract

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 pell....

Results

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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