The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.
Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.
The olefin ethylene (C2H4) was first discovered as a plant hormone in 1901 when it was observed that pea seedlings, grown in a laboratory that used coal gas lamps, exhibited an abnormal morphology in which stems (hypocotyls) were shorter, thicker and bent sideways compared to normal pea seedlings; a phenotype later termed the triple response1,2. Subsequent studies demonstrated that ethylene is a vital phytohormone that regulates numerous developmental processes such as growth, stress response, fruit ripening and senescence3. Arabidopsis thaliana, a model organism for plant biology research, has been well studied in regards to its response to ethylene. Several ethylene response mutants have been isolated by exploiting the triple response phenotype observed in dark-grown A. thaliana seedlings in the presence of ethylene1,4,5. The biosynthetic precursor for ethylene production in plants is 1-aminocyclopropane carboxylic acid (ACC)6 and is commonly used during the triple response assay to increase endogenous ethylene production that leads to the triple response phenotype1,4,5.
Although the ethylene response is widely studied in plants, the effect of exogenous ethylene on bacteria is vastly understudied despite the close association of bacteria with plants. One study reported that certain Pseudomonas strains can survive using ethylene as a sole source of carbon and energy7. However, only two studies have demonstrated that bacteria respond to ethylene. The first study showed that strains of Pseudomonas aeruginosa, P. fluorescens, P. putida, and P. syringae were chemotactic toward ethylene using an agarose plug assay in which molten agarose was mixed with a chemotaxis buffer equilibrated with pure ethylene gas8. However, to our knowledge, there have been no further reports using pure ethylene gas to characterize bacterial ethylene response, likely due to the difficultly of handling gases in the laboratory without specialized equipment. The second report of bacterial ethylene response demonstrated that ethylene increased bacterial cellulose production and influenced gene expression in the fruit-associated bacterium, Komagataeibacter (formerly Gluconacetobacter) xylinus9. In this case, the ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA) was used to produce ethylene in situ within the bacterial growth medium, bypassing the need for pure ethylene gas or specialized equipment.
CEPA produces ethylene at a 1:1 molar ratio above pH 3.510,11 through a base-catalyzed, first-order reaction12–14. The degradation of CEPA is positively correlated with pH and temperature13,14 and results in the production of ethylene, chloride and phosphate. CEPA provides researchers interested in studying bacterial responses to ethylene with a convenient alternative to gaseous ethylene.
The overall goal of the following protocols is to provide a simple and efficient method to study bacterial ethylene response and includes validation of physiologically relevant levels of ethylene production from CEPA decomposition in bacterial growth medium, analysis of culture pH to ensure CEPA decomposition is not impaired during bacterial growth, and assessment of the effect of ethylene on bacterial morphology and phenotype. We demonstrate these protocols using K. xylinus, however, these protocols can be adapted to study ethylene response in other bacteria by using the appropriate growth medium and phenotype analyses.
1. Chemicals
2. Verifying Ethylene Production from 2-Chloroethylphosphonic Acid Decomposition: Triple Response Assay
Figure 1: Setup of agar plates used for the triple response assay with CEPA. A schematic illustrates the quadrants specific for the negative control (A), positive control (B), and experimental plates (C). This figure has been modified from Augimeri and Strap9. Please click here to view a larger version of this figure.
3. Analysis of pH throughout Bacterial Growth
4. Colony Morphology
5. Pellicle Assays
Figure 2: Flow-chart illustrating the protocol used for pellicle assay and analysis. Stock CEPA-supplemented pH 7 SH medium (60 ml) is aliquoted for three separate biological replicate inoculations and a sterile control (14 ml each). These cultures are then aliquoted into six technical replicates (2 ml) into a 24 well plate and then sealed with paraffin film. After incubation for 7 days at 30 °C, pellicles are harvested and characterized by determining wet weight, thickness, dry weight, and crystallinity by FT-IR. Please click here to view a larger version of this figure
A schematic plate setup for verification of ethylene liberation from CEPA in SH medium (pH 7) by the triple response assay is shown in Figure 1A–C. A flow-chart illustrating the pellicle protocol is shown in Figure 2. Dark-grown A. thaliana seedlings exhibit the triple response phenotype (shorter and thicker hypocotyl with an exaggerated apical hook) in the presence of ACC and in the presence of ethylene produced through the decomposition of CEPA on SH medium (pH 7), but not under untreated conditions (Figure 3A)9. The hypocotyl length of ACC- and CEPA-derived ethylene treated A. thaliana seeds were significantly (p < 0.0001) shorter than untreated controls (Figure 3B)9, confirming that ethylene was released from CEPA on SH medium (pH 7) at a physiologically relevant concentration. The pH of untreated and CEPA-treated K. xylinus cultures remained above 5 (Figure 4)9; therefore CEPA decomposition into ethylene was not impaired due to bacterial organic acid production. CEPA-derived ethylene increased bacterial cellulose production when K. xylinus was grown on solid SH medium (pH 7) (Figure 5)9. All concentrations of CEPA-derived ethylene significantly decreased pellicle wet weight (Figure 6A), did not affect pellicle thickness (Figure 6B) and significantly increased pellicle dry weight (cellulose yield; Figure 6C)9. A representative photo taken for pellicle thickness measurement is shown in Figure 7. Pellicle hydration was reduced by all concentrations of CEPA-derived ethylene (Figure 8A)9, while all concentrations of CEPA-derived ethylene9 increased pellicle crystallinity (Figure 8B). The effects of NaCl and NaH2PO4 were insignificant in all cases (data not shown) confirming that observed phenotypes were caused by CEPA-derived ethylene.
Figure 3: CEPA decomposes on SH medium (pH 7) to produce ethylene. Digital USB microscope photographs show that dark-grown A. thaliana seedlings display the triple response phenotype (shorter hypocotyl, thicker hypocotyl and exaggerated apical hook) when grown in the presence of ACC and CEPA-derived ethylene compared to the untreated control (A). The hypocotyl length of seedlings grown in the presence of ACC and CEPA-derived ethylene were significantly shorter than the untreated control (B). Scale bar = 1 mm. Error bars show SD (n = 3). Bars with different letters are significantly different (p < 0.0001). This figure has been modified from Augimeri and Strap9. Please click here to view a larger version of this figure.
Figure 4: The pH of K. xylinus cultures remains high enough for CEPA decomposition in SH medium (pH 7). The culture pH stays above 5, allowing for efficient decomposition of CEPA into ethylene throughout the bacterial growth cycle. The pH of K. xylinus cultures must be monitored as they secrete and resorb organic acids. The legend shows the CEPA concentrations tested. Note that the y-axis begins at pH 5. Error bars show SD (n = 3). This figure has been modified from Augimeri and Strap9. Please click here to view a larger version of this figure.
Figure 5: CEPA-derived ethylene increases cellulose production by K. xylinus on a solid medium. K. xylinus was grown on SH agar plates (pH 7) that were untreated (A), or pre-treated with acidified (pH 2.5) ultra-pure water (B), phosphate and chloride (C–E) or CEPA (F–H). Representative colonies are shown. The arrow shows cellulose produced by K. xylinus, seen as the hazy substance around the colony. Scale bar = 0.5 mm. This figure has been modified from Augimeri and Strap9. Please click here to view a larger version of this figure.
Figure 6: CEPA-derived ethylene decreases the water-holding capacity and increases the yield of K. xylinus cellulose pellicles. Cultures were grown statically in SH broth (pH 7) supplemented with CEPA in 24-well plates, and incubated at 30 °C for 7 days before pellicles were harvested and analyzed. CEPA-derived ethylene decreased pellicle wet weight (A), had no effect on pellicle thickness (B) and increased pellicle dry weight (C). The different CEPA treatments were not significantly different from each other. Error bars show SD (n = 3). Bars with different letters are significantly different (p < 0.05). This figure has been modified from Augimeri and Strap9. Please click here to view a larger version of this figure.
Figure 7: Representative photograph of K. xylinus pellicles. Pellicle thickness was measured from photographs using ImageJ software. Scale bar = 10 mm. Please click here to view a larger version of this figure.
Figure 8: CEPA-derived ethylene reduced the hydration of K. xylinus cellulose pellicles by increasing pellicle crystallinity. Cultures were grown statically in SH broth (pH 7) in 24-well plates, and incubated at 30 °C for 7 days before pellicles were harvested and analyzed. Pellicle hydration was calculated as the difference between pellicle wet and dry weight. CEPA-derived ethylene reduced pellicle hydration (A) by increasing pellicle crystallinity (B). The different CEPA treatments were not significantly different from each other. Error bars show SD (n = 3). Bars with different letters are significantly different (p < 0.05). This figure has been modified from Augimeri and Strap9. Please click here to view a larger version of this figure.
The methods described here outline the in situ production of ethylene from CEPA for the study of bacterial ethylene response using the model organism, K. xylinus. This method is very useful as ethylene can be produced by supplementing any aqueous medium that has a pH greater than 3.510,11 with CEPA negating the need for pure ethylene gas or specialized laboratory equipment. This method is not limited to studying the effects of CEPA-derived ethylene on bacteria but can be also be adapted to study ethylene response in eukaryotic organisms. It is important that parallel control experiments be performed with phosphate and chloride as these compounds are also produced from CEPA decomposition in situ. Another necessary control is to ascertain whether CEPA itself exerts a direct effect on the microbial cultures under investigation. Physiologically irrelevant ethylene concentrations were produced when K. xylinus cultures were grown in a pH 5 SH medium and therefore serve as a control for CEPA effects9; however, this approach only works for acid tolerant organisms.
The use of CEPA-derived ethylene for biological studies in an aqueous growth medium requires verification that ethylene is indeed being produced within the system. The method described here utilizes the A. thaliana triple response assay which exploits the triple response phenotype exhibited by dark-grown seedlings in the presence of ethylene1. Petri dishes divided into four quadrants allow the seedlings to be grown in MS medium separately, but adjacent to the relevant microbial growth medium. Application of CEPA solution onto the microbial growth medium facilitates ethylene production and exposes the adjacent seedlings to an exogenous source of ethylene as it infiltrates the headspace. Supplementing the MS medium with ACC provides a positive control as it enhances the endogenous production of ethylene by A. thaliana and subsequent induction of the triple response phenotype.
Culture pH is another important consideration when producing ethylene from CEPA in microbial growth medium13,14. The SH medium typically used to culture K. xylinus has a pH of about 5 which substantially reduces the production of CEPA-derived ethylene (Augimeri and Strap, unpublished data); therefore, the medium was adjusted to pH 79. This is consistent with other studies that demonstrated that the rate of CEPA breakdown is extremely slow at or below pH 5, while it is greatly enhanced at pH 713. It is therefore essential to ensure that the growth medium of the test organism remains above pH 5. K. xylinus cultures reached a minimum pH of approximately 5.5, allowing for evolution of ethylene9. It is important to note that the absolute concentration of ethylene cannot be controlled under these culture conditions.
The effect of CEPA-derived ethylene on the growth, cellulose production, and pellicle properties of K. xylinus was used to illustrate that bacterial ethylene responses could be examined using CEPA. Adapting these methods to study ethylene response in other organisms is as simple as changing the growth medium and phenotype analyses. The use of CEPA provides researchers with a convenient substitute for ethylene gas and may encourage more ethylene-mediated studies in bacteria.
The authors have nothing to disclose.
The authors thank Dr. Dario Bonetta for providing Arabidopsis thaliana seeds and for technical assistance in regards to the triple response assay, as well as Simone Quaranta for help with FT-IR. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (NSERC-DG) to JLS, an Ontario Graduate Scholarship (OGS) to RVA, and a Queen Elizabeth II Graduate Scholarship in Science and Technology (QEII-GSST) to AJV.
1-aminocyclopropane carboxylic acid (ACC) | Sigma | A3903 | Biosynthetic precursor of ethylene in plants |
4-sector Petri dish | Phoenix Biomedical | CA73370-022 | For testing triple response |
Agar | BioShop | AGR001.1 | To solidify medium |
Canon Rebel T1i DLSR camera | Canon | 3818B004 | For pictures of pellicles |
Cellulase from Trichoderma reesei ATCC 26921 | Sigma | C2730 | Aqueous solution |
Citric acid | BioShop | CIT002.500 | For SH medium |
Commercial bleach | Life Brand | 57800861874 | Bleach for seed sterilization |
Concentrated HCl | BioShop | HCL666.500 | Hydrochloric acid for pH adjustment |
Digital USB microscope | Plugable | N/A | For pictures of colonies |
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 |
Microcentrifuge tube | LifeGene | LMCT1.7B | 1.7 mL microcentrifuge tube |
Murashige and Skoog (MS) basal medium | Sigma | M5519 | Arabidopsis thaliana growth medium |
Na2HPO4·7H2O | BioShop | SPD579.500 | Sodium phosphate, dibasic heptahydrate for SH medium |
NaCl | BioBasic | SOD001.1 | Sodium chloride for saline and control solution |
NaH2PO4·H2O | 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 |
Sucrose | BioShop | SUC600.1 | Sucrose for MS medium |
Yeast extract | BioBasic | G0961 | For SH medium |