Name: quantifying bacterial surface swarming motility on inducer gradient plates
Uploaded: 2022-01-05T13:00:00
Description: Watch this Scientific Journal Video about Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates at JoVE.com

The development of this technique was supported by the funds from the Ministry of Science and Technology&#39;s National Key R&#38;D plan (2018YFA0902604), National Natural Science Foundation of China&#39;s Research Fund for International Young Scientists (22050410270) and Shenzhen Institutes of Advanced Technology External Funds (DWKF20190001). We would like to offer our sincere gratitude to Miss Chen Xinyi for her assistance in proofreading the document and laboratory management.

1. Preparation of gradient swarm plates
<ol>
	<li>Preparation of swarm medium 
	NOTE: See the discussion section for a brief comparison of different medium viscosities; 0.7% (w/v) agar concentration of swarm medium was used in this protocol.
	<ol>
		<li>Prepare Lysogeny broth (LB) powder with agar in two conical flasks; each flask contains 2 g of tryptone, 2 g of sodium chloride, 1 g of yeast extract, and 1.4 g of agar. Add double-distilled water (ddH2O) and stir the suspension using a ...

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+swarming+and+virulence+factor+expression+in+Proteus+mirabilis+by+resveratrol.">Inhibition of swarming and virulence factor expression in Proteus mirabilis by resveratrol.</a> Journal of Medical Microbiology. 55, 1313-1321 (2006).</li><li>Zahringer, F., Massa, C., Schirmer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+enzymatic+production+of+the+bacterial+second+messenger+c-di-GMP+by+the+diguanylate+cyclase+YdeH+from+E.+coli.">Efficient enzymatic production of the bacterial second messenger c-di-GMP by the diguanylate cyclase YdeH from E. coli.</a> Applied Biochemistry and Biotechnology. 163 (1), 71-79 (2011).</li><li>Kuchma, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+di-GMP-mediated+repression+of+swarming+motility+by+Pseudomonas+aeruginosa+PA14+requires+the+MotAB+stator.">Cyclic di-GMP-mediated repression of swarming motility by Pseudomonas aeruginosa PA14 requires the MotAB stator.</a> Journal of Bacteriology. 197 (3), 420-430 (2015).</li><li>Heering, J., Alvarado, A., Ringgaard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cellular+differentiation+and+single+cell+imaging+of+Vibrio+parahaemolyticus+swimmer+and+swarmer+cells.">Induction of cellular differentiation and single cell imaging of Vibrio parahaemolyticus swimmer and swarmer cells.</a> Journal of Visualized Experiments: JoVE. (123), e55842 (2017).</li><li>Bru, J. L., Siryaporn, A., H&#248;yland-Kroghsbo, N. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swarming+of+Pseudomonas+aeruginosa+is+controlled+by+a+broad+spectrum+of+transcriptional+regulators,+including+MetR.">Swarming of Pseudomonas aeruginosa is controlled by a broad spectrum of transcriptional regulators, including MetR.</a> Journal of Bacteriology. 191 (18), 5592-5602 (2009).</li><li>Delprato, A. M., Samadani, A., Kudrolli, A., Tsimring, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swarming+ring+patterns+in+bacterial+colonies+exposed+to+ultraviolet+radiation.">Swarming ring patterns in bacterial colonies exposed to ultraviolet radiation.</a> Physical Review Letters. 87 (15), 158102 (2001).</li><li>Araujo Neto, L. A., Pereira, T. M., Silva, L. 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Investigation of bacterial swarming motility on semisolid gradient plates can be a challenging task18,19,20, as it involves multiple factors such as substrate viscosity, humidity, and medium components. Among these factors, agar concentration plays a decisive role in determining microbial reversion to either swimming or swarming motility. As the agar concentration increases from 0.3% (w/v) to 1% (w/v), bacterial swimming motilit...

Bacterial swarming motility refers to the collective migration of bacterial cells across the surface of a substance. In addition to semisolid agar plates specially prepared in the laboratory1, this phenotype is also observed on some soft substrates such as animal tissues2, hydrated surfaces3, and plant roots4. While a semisolid surface is considered one of the fundamental conditions for bacterial swarming, some species also require an energy-rich medium to support their swarming motility5. Flagella rotation powers both swimming and swarming motility-swimming describes the unicellular motility within a liquid environment, whereas swarming is the synchronous movement of a microbial population across semisolid surfaces.
Substrate viscosity influences bacterial motility; studies of pathogenic microbes, such as Helicobacter pylori,&#160;have shown that the pathogen&#39;s motility changes depending on the mucin layer viscosity, which is influenced by environmental acidification in the human host6. To replicate these environments, earlier studies using agar concentration above 0.3% (w/v) restrict bacterial swimming motility to effect a gradual shift into surface swarming. The use of agar concentration above 1% (w/v) prevents the swarming motility of many species7. The colony patterns formed on the surface are diverse, including featureless mat8, bull&#39;s eye9, dendrites10, and vortex11.
Although the relevance of such patterns remains unclear, those patterns seem to be dependent on environmental and chemical cues12. Environmental cues cover different aspects, including temperature, salinity, light, and pH, whereas chemical cues include the presence of microbial quorum sensing molecules, biochemical byproducts, and nutrients. Autoinducer quorum sensing signaling molecules such as AHL (N-hexanoyl-L homoserine lactone) can impact surface swarming by regulating the production of surfactant13,14. Resveratrol, a phytoalexin compound, could restrict bacterial swarming motility15.
In the present work, we investigate the effect of gradient concentrations of resveratrol on wild-type Escherichia coli K12 strain and investigate arabinose-inducible swarming motility of engineered E. coli K12-YdeH and Pseudomonas aeruginosa PAO1-YdeH species. The production of the YdeH enzyme is induced by arabinose via the araBAD promoter, resulting in cellular c-di-GMP perturbation and affecting bacterial swarming motility16,17. This inducible swarming behavior is studied using arabinose gradient swarm plates with E. coli K12-YdeH and P. aeruginosa PAO1-YdeH strains.
The gradient swarm plates are prepared by successively solidifying double-layer medium (Figure 1B). The bottom layer comprises the medium added with the inducer, poured on one side of a propped-up Petri dish. Upon the solidification of the bottom layer, the Petri dish is returned to a flat surface, where the upper layer containing the medium without the inducer is added from the other side of the plate. After the swarm plates are completely solidified, isometrically arranged holding-wells are produced by boring holes on the swarm plates following a fixed layout (Figure 1C) or by imprinting the wells using a 3D printed model of the plate cover containing pegs during the medium curing process (Supplemental Figure S1). A gel imaging system is used to capture the swarming morphologies at different time points (Figure 2). Analysis of surface swarming using ImageJ software (Supplemental Figure S2) provides quantitative results of the surface swarming process (Figure 3).
Thus, we propose a simple method to test surface swarming motility within a concentration range of inducers. This method can be used to test multiple concentration responses of other inducers by mixing the inducer into the bottom-layer medium and can be applied to other motile species (e.g., Bacillus subtilis, Salmonella enterica, Proteus mirabilis, Yersinia enterocolitica). This approach could provide reliable qualitative and quantitative results for screening a single chemical inducer, and separate plates may be employed to evaluate more chemical inducers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>V900500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Solarbio</td>
			<td>A8180</td>
			<td>25 g, &#8805; 85% (GC)</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Corning</td>
			<td>430790</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>Cryogenic vial</td>
			<td>Corning</td>
			<td>430488</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Aladdin</td>
			<td>D103272</td>
			<td>AR, &#62; 99% (GC)</td>
		</tr>
		<tr>
			<td>L(+)-Arabinose</td>
			<td>Aladdin</td>
			<td>A106195</td>
			<td>98% (GC), 500 g</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Bkman</td>
			<td>B-SLPYM90-15</td>
			<td>Plastic Petri dishes,circular,90 mm x 15 mm</td>
		</tr>
		<tr>
			<td>Resveratrol</td>
			<td>Aladdin</td>
			<td>R107315</td>
			<td>99% (GC), 25 g</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Macklin</td>
			<td>S805275</td>
			<td>AR, 99.5% (GC), 500 g</td>
		</tr>
		<tr>
			<td>Square Petri dishes</td>
			<td>Bkman</td>
			<td>B-SLPYM130F</td>
			<td>Plastic Petri dishes, square, 13 mm x 13 mm</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Scientific Oxoid</td>
			<td>LP0042</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Thermo Scientific Oxoid</td>
			<td>LP0021</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biochemical incubator</td>
			<td>Blue pard</td>
			<td>LRH-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Tanon 5200multi imaging system</td>
			<td>Tanon</td>
			<td>5200CE</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic water bath</td>
			<td>Jinghong</td>
			<td>DK-S28</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying bacterial surface swarming motility on inducer gradient plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

The workflow consisting of the preparation of gradient swarm plates, inoculation, and incubation is shown in Figure 1B. To generate gradient swam plates, the bottom-layer medium is poured into propped-up dishes at ~4.3&#176; from the horizontal plane (Supplemental Figure S3), followed by pouring the upper-layer medium after the bottom layer is completely solidified. The composition of the double-layer medium is shown in Table 1. Then, bacterial culture cultu...

Watch this Scientific Journal Video about Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates at JoVE.com

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

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