Name: High-throughput Method for Observing Motility Phenotypes in Pseudomonas aeruginosa (Video) | JoVE Journal
Uploaded: 2025-06-20T12:30:00.000+00:00
Duration: 7 min 23 s
Description: Learn a high-throughput method for observing motility phenotypes in Pseudomonas aeruginosa using transposon mutant libraries and image analysis. This protocol enables genome-wide identification of genetic factors controlling swarming and twitching behaviors.

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5 minutes

We thank the Surette lab at McMaster University for providing a copy of the PA14 library. We thank Dr. Fabrice Jean-Pierre for his helpful comments on the manuscript. This work was supported by the Natural Science and Engineering Research Council of Canada (RGPIN-2019-06044) and a starting grant for new investigators from the Fonds de recherche du Qu&#233;bec - Sant&#233; (FRQS; #295613). J.-P.C. holds a Chercheur boursier junior 2 fellowship from the Fonds de recherche du Qu&#233;bec-Sant&#233; (FRQS).

NOTE: The general workflow of this procedure is outlined in Figure 1.
<img alt="Motility assay workflow: P. aeruginosa transposon library, 96/384 plates, phenotype analysis." class="xfigimg" src="/files/ftp_upload/67761/67761fig01.jpg" data-altupdatedat="1773047343000" data-alt="Figure 1"> 
Figure 1: Experimental workflow. (1) Prepare the necessary quantity of motility media based on the type of motility of interest in the experiments. (2) Using a manual or automated replicator system, prepare the source stock plates for the P. aeruginosa transposon insertion mutant library of the mutant collection at the appropriate density, with emphasis on precision and uniform inoculation across all wells of the plates. (3) Transfer samples from the source plates of the mutant collection to the motility plates. (4) After incubation, observe and analyze motility phenotypes and identify the genes involved in the motility of interest using high quality images. (5) Perform traditional motility assays to validate the hits identified through high-throughput motility experiments. Created with BioRender. <a href="https://www.jove.com/files/ftp_upload/67761/67761fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Swarming plates - M9 media</td>
			<td>Twitching plates - LB media</td>
			<td>Source plates - LB media</td>
		</tr>
		<tr>
			<td>200 mL of a 5x M9 salts solution</td>
			<td>10 g NaCl </td>
			<td>10 g NaCl </td>
		</tr>
		<tr>
			<td>10 mL of 20% glucose</td>
			<td>10 g Tryptone </td>
			<td>10 g Tryptone </td>
		</tr>
		<tr>
			<td>25 mL of casamino acids</td>
			<td>5 g Yeast Extract </td>
			<td>5 g Yeast Extract </td>
		</tr>
		<tr>
			<td>1 mL of 1M MgSO4</td>
			<td>10 g Agar </td>
			<td>15 g Agar </td>
		</tr>
		<tr>
			<td>500 mL of 1% agar</td>
			<td>Add dH2O to 1L</td>
			<td>Add dH2O to 1L</td>
		</tr>
		<tr>
			<td>Add dH2O to 1L</td>
			<td></td>
			<td>Gentamicin (final concentration 15 µg/mL)</td>
		</tr>
	</tbody>
</table>
Table 1: Media composition for the various plates used in the assay.
1. Preparing culture medium plates
NOTE: See Table of Materials to prepare stock solutions and storage conditions. Plates should be filled on a leveled surface. When drying, plates should be flat on the surface (i.e., not stacked). This ensures that the culture medium in the plate dries evenly. Any uniwell plates with a typical footprint can work, but plates with larger internal dimensions (without inside walls), such as the Singer Instruments Plus Plates or VWR single well non-treated tissue culture plates, facilitate working with a higher density of colonies per plate.
<ol>
	<li>Prepare an appropriate quantity of media for the experiments as per the recipe in Table 1. Keep the media at ~45 ˚C- 50 ˚C to prevent the agar from solidifying.
	<ol>
		<li>For source plates, prepare 59 LB (lysogeny broth) plates containing 1.5% agar and 15 µg/mL of gentamycin, which is needed to replicate the complete PA14 library-59 x 25 mL =~ 1500 mL. Prepare another 15 plates of LB 1.5% agar and 15 µg/mL gentamycin to upscale the library in 384-density format-15 x 25 mL = ~400 mL.</li>
		<li>Twitching plates: If working in 96-density format, prepare 59 twitching plates (LB 1% agar). Prepare 15 twitching plates if working in 384-density format.</li>
		<li>Swarming plates: Prepare 15 (384-density format) or 59 (96-density format) swarming plates (M9-glucose 0.5% agar)</li>
	</ol>
	</li>
	<li>Take the lid off a plate and pour 25 mL of media per plate. Distribute the media evenly across the plate by gently lifting every corner of the plate one at a time. Repeat this wiggle motion 2x-3x. Put the lid back on the plate.</li>
	<li>Let the plates solidify and dry at room temperature (RT) overnight. If the plates are not used immediately, put them in hermetic bags and store them in a dry place. They can be stored for 3 weeks.</li>
</ol>
2. Preparation of source plates of the P. aeruginosa transposon mutant library
NOTE: Source plates refer to LBA-gentamycin plates containing a copy of the PA14 library that are used later to inoculate the motility plates. The P. aeruginosa transposon mutant library (PA14 library) is a set of 59 frozen 96-well plates29. Steps to prepare source plates for each format are described hereafter. This protocol is adaptable for laboratories that do not have access to the Rotor+ replicator. Researchers can successfully perform the same experimental procedures using manual replicators, such as the spring-loaded 96-pin replicators used here for the replication of the frozen plates of the PA14 library during Day 1 (refer to the Table of Materials) or any other 96-pin replicators. Manual replicators need to be sterilized and allowed to cool down between each use.
<ol>
	<li>Day 1: Replication of the PA14 library from the 59 frozen 96-well plates onto source plates
	<ol>
		<li>Sterilize a spring-loaded 96-pin replicator by placing it on a hot plate at max temperature for 8 min. After sterilization, allow the replicator to cool sufficiently (~10 min) to avoid killing bacteria.</li>
		<li>Grab one source plate. Using the sterile 96-pin replicator, carefully dip the replicator pins into one 96-well plate, then transfer the cells directly onto the surface of the source plate. Repeat until all 59 frozen 96-well plates of the PA14 library are replicated onto source plates.</li>
		<li>Incubate the 96-density source plates at 37 °C for 16-18 h. 
		NOTE: If using the method with a 96-density format, these source plates can then be used to inoculate the motility plates.</li>
	</ol>
	</li>
	<li>Day 2: Upscaling from 96-density format to 384-density format 
	NOTE: Day 2 can be skipped if working at 96-density.
	<ol>
		<li>After the incubation, leave the plates (flat on a surface, not stacked) at room temperature (~1 h) to cool down. If there is condensation on the lids, remove the condensation using a delicate task wipe to prevent water droplets from falling onto the plates, which could cause cross-contamination between colonies.</li>
		<li>Configure the replicator with the following parameters to ensure accurate colony transfer. Turn ON the replicator. In the section Select an operate mode, select Select and Run Stored Programs.</li>
		<li>In the section Select source plates, select PlusPlate 96 Agar. In the section Select target plates, select PlusPlate 384 Agar. In the section Select pads, select Short Pin 96.</li>
		<li>In the section Replicate program options, select General and click on Recycle &gt; None.</li>
		<li>In the Source section, select Offset and click on Random &gt; Default Radius. In the section Target, select Pinning and adjust the pressure to 20%. Leave all other settings as default.</li>
		<li>Insert the Short Pin RePads 96 into the appropriate compartment.</li>
		<li>Position the source and target plates on the designated platform of the replicator. Place the 96-density source plates (plates 1, 2, 3, and 4) and the new empty source plate (LBA plates) onto the platform. Using the selected parameters, the microbial array pinning robot will transfer colonies from the 96-density plates to the new 384-density plate. Press Run.</li>
		<li>Change all plates on the platform (the next four 96-density source plates and a new empty source plate) and repeat until all plates have been upscaled. For the last set of plates, use plate 57, 58, 59, and 1. Each 384-density plate will consist of alternating colonies from four 96-density plates (see Figure 2). Incubate the 384-density LBA plates at 37 °C for 16-18 h. 
		NOTE: These 384-density source plates can then be used to inoculate the motility plate to perform the protocol at 384-density. At any step after incubation, plates can be stored at 4 °C for up to 3 weeks.</li>
	</ol>
	</li>
</ol>
<img alt="Hierarchical clustering diagram illustrating color-coded data grouping." class="xfigimg" src="/files/ftp_upload/67761/67761fig02.jpg" data-altupdatedat="1773047343000" data-alt="Figure 2"> 
Figure 2: Upscaling from 96-density plates to 384-density plates. Colonies from four 96-density source plates are transferred and merged into a single 384-density plate. Each quadrant of the 384-density plate corresponds to one of the original 96-well plates (red, blue, yellow, and green), preserving the position of each mutant. Created with BioRender. <a href="https://www.jove.com/files/ftp_upload/67761/67761fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. High-throughput motility experiment
NOTE: The protocol was performed here in 384-density format, but the same protocol can be adapted to a 96-density format by modifying the parameters on the replicator to transfer from source plates to motility plates using the 96 selection instead of 384.
<ol>
	<li>After the incubation, leave the plates (flat on a surface, not stacked) at room temperature (~1 h) to cool down. If condensation on the lids occurs, remove it using a delicate task wipe to prevent water droplets from falling onto the plates, which could cause cross-contamination between colonies.</li>
	<li>Grab the appropriate number of plates (59 for a 96-density format or 15 for a 384-density format) for the desired assay: M9-glucose 0.5% agar plates for swarming, LB, and 1% agar plates for twitching.</li>
	<li>Configure the replicator with the following parameters to ensure accurate colony transfer.
	<ol>
		<li>Turn ON the replicator. In the section Select an operate mode, select Select and Run Stored Programs. In the section Select source plates, Select PlusPlate 384 Agar.</li>
		<li>In the section Select target plates, select PlusPlate 384 Agar. In the section Select pads, select Short Pin 384. In the section Replicate program options, select General &gt; Recycle &gt; None.</li>
		<li>In Singer Programs, select Replicate Many.</li>
		<li>In the section Source, select Offset &gt; Random &gt; Default Radius. In the section Target, select Pinning and adjust the pressure to 2%. Leave all other settings as default.</li>
	</ol>
	</li>
	<li>Insert the Short Pin RePads 384 in the appropriate compartment.</li>
	<li>Position the source and target plates on the designated platform of the replicator. Place the 384-density source plates and the empty motility plates onto the platform. Press Run Using the Selected Parameters, the microbial array pinning robot will transfer colonies from the 384-density source plates to the motility plates. Each 384-density plate will consist of a copy from one 384-density source plate.</li>
	<li>Ensure that the transfer is even and that all colonies are appropriately pinned onto the agar surface of the motility plates. Put the inoculated motility plates in plastic bags. Carefully place the bags containing the plates into an incubator set to 37 °C for 18 h. 
	NOTE: The plates are placed in bags to maintain humidity and prevent uneven drying. Place the plates in the incubator with the lids facing up.</li>
	<li>After incubation, check the motility plates for even colony growth and absence of contamination.</li>
	<li>Day 3: Imaging motility plates 
	NOTE: The Singer Instruments PhenoBooth is used to capture high-resolution images of the motility plates. It uses a scientific-grade camera and produces 23-megapixel images. It includes five lighting channels to capture high-resolution images of colonies from Petri dishes and rectangular plates. Other high-quality imagers also work.
	<ol>
		<li>After the incubation period, the motility plates are ready for the imaging process. Turn ON the Phenobooth and click on the Icon corresponding to the Phenobooth software.</li>
		<li>A Select Project page opens; click on New Project &gt; Colony Counting. Choose the Desired File Path to store the images in the Location field. In the Resolution field, choose 5626 x 4220. Click OK, the device is now ready to take pictures.</li>
		<li>Insert the plate into the reader with the medium side up after removing the lid. Ensure the plate is properly positioned before closing the reader to avoid jamming the plate.</li>
		<li>Set the parameter to white light in the Lighting tab. Click on Preview. Adjust the exposure settings in the PhenoBooth settings tab to obtain the optimal parameters for observing motility phenotypes.</li>
		<li>Click on Acquire; the captured images will appear on the left side of the screen. Once finished, close the software (all captured images will be saved in the previously chosen file in the Location tab). 
		NOTE: Phenobooth saves images in JPEG format, but for the downstream analysis steps, images can be in TIFF, JPEG, or PNG format.</li>
	</ol>
	</li>
</ol>
4. Analysis of motility phenotypes
NOTE: Image analysis is performed as described in detail by French et al.30,31. A script to perform data analysis is available from31(ImageJ Macro).
<ol>
	<li>Briefly, images are processed in ImageJ32, which generates a value for each colony on the motility plate. First, convert images to 8-bit format, and segment the colonies and their associated motility areas from the background using the threshold command. Draw a Region of Interest (ROI) around every colony and measure the motility area. This generates a table containing an integrated density value for each well, organized by rows. This table can be saved as a CSV file for further analysis.
	<ol>
		<li>Run the macro in ImageJ and open the folder containing the images.</li>
		<li>When prompted, adjust the rotation of the plate by modifying the angle of the plate using the Preview option so that the plate is straight. When satisfied, click the OK button.</li>
		<li>Next, move the rectangle around the colonies and use it to crop the portion of the plate containing the colonies. When ready, click the OK button.</li>
		<li>The next prompt will define the grid used to draw ROI around each colony and measure the motility area. Select the appropriate colony density. Adjust the various parameters if needed, so that each circle is around a colony. When ready, activate the Okay? box and click the OK button. This will measure the motility area for each colony and save a CSV file into the folder containing the data.</li>
	</ol>
	</li>
	<li>Combine this CSV file with the plate legend, also organized by rows, to identify each motility area with the corresponding mutant. Lower integrated density values correspond to the mutants that were non-motile or showed decreased motility.</li>
</ol>
5. Traditional motility assays for validation of hits
NOTE: After observing mutants with impacted motility in the high-throughput motility protocol, it is important to validate the obtained hits individually. The individual motility protocol differs from the one used for high-throughput testing, but the media used are the same.
<ol>
	<li>Swarming motility assay 
	NOTE: Swarming motility is performed as described in detail by Ha et al.16.
	<ol>
		<li>Briefly, autoclave an agar solution with M9 (0.5% agar), glucose, casamino acids, and MgSO4. Pour the cooled, mixed agar into Petri plates (25 mL per plate).</li>
		<li>Inoculate with 2.5 µL of overnight bacterial culture. Incubate at 37 °C for 18 h with the lids facing up to observe motility phenotypes.</li>
	</ol>
	</li>
	<li>Twitching motility assay 
	NOTE: Twitching motility is performed as described in detail by Haley et al.33,34.
	<ol>
		<li>Briefly, autoclave a LB 1% agar (see Table 1). Pour the cooled, mixed LB 1% agar into Petri plates (25 mL per plate).</li>
		<li>Stab-inoculate bacteria to the bottom of twitching plates (1% agar). Incubate the plates at 37 °C for 48 h and then at RT for an additional 48 h. Remove the agar from the plate and visualize the twitching zone by staining the Petri dish with 1% crystal violet.</li>
		<li>Photograph the Petri dishes using the PhenoBooth with the same parameters described in step 3.</li>
	</ol>
	</li>
</ol>

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<li>Tong, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+dispensability+in+Escherichia+coli+grown+in+thirty+different+carbon+environments%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">Gene dispensability in Escherichia coli grown in thirty different carbon environments.</a> mBio. 11 (6), e02259-e02320 (2020).</li>
<li>French, S., Côté, J. P., Stokes, J. M., Truant, R., Brown, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacteria+getting+into+shape%3A+genetic+determinants+of+E.+coli+morphology%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">Bacteria getting into shape: genetic determinants of E. coli morphology.</a> mBio. 8, e01977-e02016 (2017).</li>
<li>Porwollik, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defined+single-gene+and+multi-gene+deletion+mutant+collections+in+Salmonella+enterica+sv+Typhimurium%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.</a> PLoS One. 9 (7), e99820(2014).</li>
<li>Koo, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction+and+analysis+of+two+genome-scale+deletion+libraries+for+Bacillus+subtilis%5BTitle%5D)+AND+%22Cell+Syst%22%5BJournal%5D)">Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis.</a> Cell Syst. 4 (3), 291-305.e7 (2017).</li>
<li>Liberati, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+ordered%2C+nonredundant+library+of+Pseudomonas+aeruginosa+strain+PA14+transposon+insertion+mutants%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants.</a> Proc Natl Acad Sci U S A. 103 (8), 2833-2838 (2006).</li>
<li>French, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+robust+platform+for+chemical+genomics+in+bacterial+systems%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">A robust platform for chemical genomics in bacterial systems.</a> Mol Biol Cell. 27 (6), 1015-1025 (2016).</li>
<li>French, S., Guo, A. B. Y., Brown, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comprehensive+guide+to+dynamic+analysis+of+microbial+gene+expression+using+the+3D-printed+PFIbox+and+a+fluorescent+reporter+library%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">A comprehensive guide to dynamic analysis of microbial gene expression using the 3D-printed PFIbox and a fluorescent reporter library.</a> Nat Protoc. 15 (2), 575-603 (2020).</li>
<li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NIH+Image+to+ImageJ%3A+25+years+of+image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li>
<li>Haley, C. L., Kruczek, C., Qaisar, U., Colmer-Hamood, J. A., Hamood, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mucin+inhibits+Pseudomonas+aeruginosa+biofilm+formation+by+significantly+enhancing+twitching+motility%5BTitle%5D)+AND+%22Can+J+Microbiol%22%5BJournal%5D)">Mucin inhibits Pseudomonas aeruginosa biofilm formation by significantly enhancing twitching motility.</a> Can J Microbiol. 60 (3), 155-166 (2014).</li>
<li>Déziel, E., Comeau, Y., Villemur, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initiation+of+biofilm+formation+by+Pseudomonas+aeruginosa+57RP+correlates+with+emergence+of+hyperpiliated+and+highly+adherent+phenotypic+variants+deficient+in+swimming%2C+swarming%2C+and+twitching+motilities%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities.</a> J Bacteriol. 183 (4), 1195-1204 (2001).</li>
<li>Gao, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((oprC+impairs+host+defense+by+increasing+the+quorum-sensing-mediated+virulence+of+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">oprC impairs host defense by increasing the quorum-sensing-mediated virulence of Pseudomonas aeruginosa.</a> Front Immunol. 11, 1696(2020).</li>
<li>Rosenau, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipase+LipC+affects+motility%2C+biofilm+formation+and+rhamnolipid+production+in+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22FEMS+Microbiol+Lett%22%5BJournal%5D)">Lipase LipC affects motility, biofilm formation and rhamnolipid production in Pseudomonas aeruginosa.</a> FEMS Microbiol Lett. 309 (1), 25-34 (2010).</li>
<li>Anyan, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+IV+pili+interactions+promote+intercellular+association+and+moderate+swarming+of+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Type IV pili interactions promote intercellular association and moderate swarming of Pseudomonas aeruginosa.</a> Proc Natl Acad Sci U S A. 111 (50), 18013-18018 (2014).</li>
<li>Burdman, S., Bahar, O., Parker, J. K., De La Fuente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+type+IV+pili+in+pathogenicity+of+plant+pathogenic+bacteria%5BTitle%5D)+AND+%22Genes%22%5BJournal%5D)">Involvement of type IV pili in pathogenicity of plant pathogenic bacteria.</a> Genes. 2 (4), 706-735 (2011).</li>
<li>Lewis, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonmotile+subpopulations+of+Pseudomonas+aeruginosa+repress+flagellar+motility+in+motile+cells+through+a+type+IV+pilus-+and+pel-dependent+mechanism%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Nonmotile subpopulations of Pseudomonas aeruginosa repress flagellar motility in motile cells through a type IV pilus- and pel-dependent mechanism.</a> J Bacteriol. 204 (5), e00528-e00621 (2022).</li>
<li>Chang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+sensing+for+biofilm+formation+in+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Front.+Microbiol%22%5BJournal%5D)">Surface sensing for biofilm formation in Pseudomonas aeruginosa.</a> Front. Microbiol. 8, 2671(2018).</li>
<li>Zegadło, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+motility+and+its+role+in+skin+and+wound+infections%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Bacterial motility and its role in skin and wound infections.</a> Int J Mol Sci. 24 (2), 1707(2023).</li>
<li>Jurado-Martín, I., Sainz-Mejías, M., McClean, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa%3A+An+audacious+pathogen+with+an+adaptable+arsenal+of+virulence+factors%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Pseudomonas aeruginosa: An audacious pathogen with an adaptable arsenal of virulence factors.</a> Int J Mol Sci. 22 (6), 3128(2021).</li>
<li>Qin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa%3A+pathogenesis%2C+virulence+factors%2C+antibiotic+resistance%2C+interaction+with+host%2C+technology+advances+and+emerging+therapeutics%5BTitle%5D)+AND+%22Sig+Transduct+Target+Ther%22%5BJournal%5D)">Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics.</a> Sig Transduct Target Ther. 7 (1), 199(2022).</li>
<li>Piskovsky, V., Oliveira, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+motility+can+govern+the+dynamics+of+antibiotic+resistance+evolution%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Bacterial motility can govern the dynamics of antibiotic resistance evolution.</a> Nat Commun. 14 (1), 5584(2023).</li>
<li>Xu, J., Koizumi, N., Nakamura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crawling+motility+on+the+host+tissue+surfaces+is+associated+with+the+pathogenicity+of+the+zoonotic+spirochete+Leptospira%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Crawling motility on the host tissue surfaces is associated with the pathogenicity of the zoonotic spirochete Leptospira.</a> Front Microbiol. 11, 1886(2020).</li>
<li>Rasmussen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+screening+assay+for+inhibitors+of+bacterial+motility+identifies+a+novel+inhibitor+of+the+Na%2B-driven+flagellar+motor+and+virulence+gene+expression+in+Vibrio+cholerae%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">A high-throughput screening assay for inhibitors of bacterial motility identifies a novel inhibitor of the Na+-driven flagellar motor and virulence gene expression in Vibrio cholerae.</a> Antimicrob Agents Chemother. 55 (9), 4134-4143 (2011).</li>
</ol>

The authors declare no disclosures.

The high-throughput motility protocol described here allows us to process and analyze the motility phenotype of many colonies with precision and reproducibility. Compared to traditional manual methods, this semi-automated approach increases the scalability and throughput of motility assays to allow the genome-wide assessment of bacterial motility. The protocol has been optimized for use with the PA14 library, but this assay could be used with any ordered library of mutants, such as the Keio collection for E. coli</em...

Pseudomonas aeruginosa is an opportunistic pathogen that can colonize diverse environments because of its remarkable metabolic versatility1,2. This versatility is crucial for the transition from a planktonic mode of growth to biofilms which contributes to its capacity to thrive in a wide range of niches ranging from water to the human body3,4,5. The transition between these modes of growth is helped by an extensive ability to move through these environments using various types of motility, including swimming, swarming, and twitching1,6. Each type of motility is mediated by distinct mechanisms and cellular structures and allows P. aeruginosa to respond to environmental cues7,8. Swimming motility is driven by a single polar flagellum and is used to move through liquid media towards nutrients, for instance9. Swarming motility is a coordinated movement facilitated by flagella and biosurfactant production and allows colonies to spread across semi-solid surfaces10,11. Finally, twitching motility is flagella independent. Instead, twitching is mediated by type IV pili (T4P) and used to move on top of surfaces12,13. Twitching motility also contributes to the early steps of biofilm formation of P. aeruginosa by providing initial surface attachment. Following initial attachment, twitching allows bacteria to move across surfaces and create microcolonies, which can then develop into mature biofilms1,4,13.
Traditionally, bacterial motility assays are carried out individually, one strain at a time, on a soft agar plate or using microscopy methods6,14,15. Semi-solid media have been used for many years in the study of bacterial motility. Nowadays, these techniques are still used to identify motility phenotypes in bacteria. For example, traditionally, swarming motility is observed by inoculating 2.5 &#181;L of a P. aeruginosa culture at the center of a Petri dish containing M9 media at 0.5%-0.8% agar concentration16. Twitching motility is usually measured by looking at the spread of the bacteria at the interface surface between agar (at 1%) and the base of a Petri dish after a stab inoculates P. aeruginosa through the culture media. A large halo of interstitial colony expansion is obtained after 48 h of incubation, whereas non-twitching strains produce no such zone of colony expansion1,17,18.
More recently, transposon mutagenesis approaches have discovered new genes involved in motility or biofilm formation in P. aeruginosa19,20,21. For example, new genes involved in twitching motility were found using a high-density transposon mutant library in P. aeruginosa19. Another useful tool in functional genomics is the use of ordered mutant collections22,23. The Keio collection24, containing single-gene deletions for all non-essential genes in Escherichia coli, is probably the best-known collection of mutants and has been used to define genes affecting various phenotypes from the growth in different media25 to cell morphology26 and more. Such collections are also available for Salmonella enterica serovar Typhimurium27, Bacillus subtilis28, P. aeruginosa29 as well as many other species. These collections are also well-suited to study motility in a genome-wide fashion, leading to the development of a high-throughput method to study the motility of P. aeruginosa. Here, the method has been tested by evaluating the contribution of mutants of P. aeruginosa to both twitching and swarming motility on high-density plates. This high-throughput method can be used for various motility phenotypes and is suitable for studying the motility of other bacteria as well.

<table><tbody><tr><td>96 well-plates&amp;nbsp;&amp;nbsp;</td><td>Corning&amp;nbsp;&amp;nbsp;</td><td>3701&amp;nbsp;</td><td></td></tr><tr><td>Agar&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;&amp;nbsp;</td><td>BP1423-2&amp;nbsp;</td><td>Final concentration of 0.5% for swarming motility or 1% for twitching motility respectively.&amp;nbsp;</td></tr><tr><td>Casamino Acids&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;&amp;nbsp;</td><td>223050&amp;nbsp;</td><td>Prepare a 20% stock in water. Filter sterilizes with a 0.22 &amp;mu;m filter and store at 4 &amp;deg;C. Final concentration 0.5% in M9 minimum medium.&amp;nbsp;</td></tr><tr><td>Crystal violet&amp;nbsp;</td><td>Bio Basic&amp;nbsp;&amp;nbsp;</td><td>CB0331</td><td>Prepare a 1% solution in water.&amp;nbsp;</td></tr><tr><td>D-Glucose&amp;nbsp;&amp;nbsp;</td><td>Bio Basic&amp;nbsp;&amp;nbsp;</td><td>GB0219&amp;nbsp;</td><td>Prepare a 20% stock (1.1 M) in water and sterilize by autoclaving. Final concentration 0.2% in M9 minimum medium.&amp;nbsp;</td></tr><tr><td>Filtropur S 0.2&amp;nbsp;</td><td>Sarstedt&amp;nbsp;</td><td>83.1826.001</td><td></td></tr><tr><td>Gentamycin sulfate&amp;nbsp;&amp;nbsp;</td><td>Bio Basic&amp;nbsp;&amp;nbsp;</td><td>GB0217&amp;nbsp;</td><td>Prepare a 30 mg/ml stock in water. Filter sterilizes with a 0.22 &amp;mu;m filter and store at 4 &amp;deg;C &amp;ndash; final concentration in plates 15 &amp;micro;g/mL..&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Inoculating Loop and Needle&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>22363597</td><td></td></tr><tr><td>M9 Minimal Salts, 5X&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>248510</td><td>Prepare a 1M stock in water and sterilize by autoclave. Final concentration 1 mM in M9 minimum medium.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Magnesium Sulfate (MgSO4)&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>M63-500</td><td>&amp;nbsp;Prepare a 1 M MgSO4 stock in water. Filter sterilizes with a 0.22 &amp;mu;m filter and store at room temperature. Final concentration&amp;nbsp;</td></tr><tr><td>PA14 Transposon Insertion Mutant Library&amp;nbsp;&amp;nbsp;</td><td></td><td>Liberati et al., 2006 (See reference 29)</td><td>59 96-well plates for the entire collection. Each well contains a single mutant in LB with 25% glycerol.&amp;nbsp;</td></tr><tr><td>Petri dish 92x16mm&amp;nbsp;</td><td>Sarstedt&amp;nbsp;</td><td>82.1473.001</td><td></td></tr><tr><td>PhenoBooth+&amp;nbsp;</td><td>Singer Instruments&amp;nbsp;</td><td>https://www.singerinstruments.com/solution/phenobooth/specification/&amp;nbsp;</td><td>Disolve 11.3 g of the powder in 200 mL of purified water. Autoclave at 121 &amp;deg;C for 15 min.&amp;nbsp;</td></tr><tr><td>Plastic Syringe 10 mL&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>14955459</td><td></td></tr><tr><td>Rotor+&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Singer Instruments&amp;nbsp;</td><td>https://www.singerinstruments.com/resource/rotor-hda/&amp;nbsp;</td><td></td></tr><tr><td>Short pin RePads 384 densities&amp;nbsp;</td><td>Singer Instruments&amp;nbsp;&amp;nbsp;</td><td>REP-004&amp;nbsp;</td><td></td></tr><tr><td>Short pin RePads 96 densities&amp;nbsp;&amp;nbsp;</td><td>Singer Instruments&amp;nbsp;&amp;nbsp;</td><td>REP-002&amp;nbsp;</td><td></td></tr><tr><td>Sodium Chloride (NaCl)&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>BP358-212&amp;nbsp;</td><td></td></tr><tr><td>Spring-loaded 96-pin replicator</td><td>EnzyScreen&amp;nbsp;&amp;nbsp;</td><td>CR1000&amp;nbsp;</td><td></td></tr><tr><td>Surgical blade stainless No. 25&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>08-918-5F</td><td></td></tr><tr><td>Tryptone&amp;nbsp;</td><td>Oxoid&amp;nbsp;&amp;nbsp;</td><td>LP0042B&amp;nbsp;</td><td></td></tr><tr><td>Uniwell plates</td><td>Singer Instruments&amp;nbsp;&amp;nbsp;</td><td>PlusPlates: PLU-003&amp;nbsp;&amp;nbsp;</td><td>Plate dimension: 54 &amp;times; 34 &amp;times; 38 cm - with no interior walls, which provide larger internal dimensions.&amp;nbsp;</td></tr><tr><td>Uniwell plates</td><td>VWR</td><td>Single well tissue culture plates: 75780-348</td><td>Same dimensions as the PlusPlates.</td></tr><tr><td>Yeast Extract&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>248510&amp;nbsp;</td><td></td></tr></tbody></table>

high-throughput method for observing motility phenotypes in pseudomonas aeruginosa

High-throughput Method for Observing Motility Phenotypes in Pseudomonas aeruginosa

Motility behaviors often play a significant role in the ability of a bacterium to exploit the resources available in its environment. This is particularly true for the versatile pathogen Pseudomonas aeruginosa, which can exhibit diverse types of motilities, including swarming and twitching, which are important pathogenic traits that contribute to surface colonization, biofilm formation, and evasion of host defenses. This manuscript presents a high-throughput motility protocol to study the motility behaviors of P. aeruginosa. The protocol allows simultaneous testing of multiple strains of P. aeruginosa from a genome-wide mutant library, for instance, to identify and analyze the genetic factors involved in its motility. The approach offers the possibility to study motility in a comprehensive manner and insights into the molecular mechanisms underlying P. aeruginosa's motility. The protocol described here can also be modified to accommodate different types of motility assays as well as other bacterial species, thus providing a powerful platform for advancing the understanding of bacterial behavior in various contexts.

We used this protocol to characterize the effect of gene inactivation on the motility of P. aeruginosa in high throughput. Here, we detailed its use to study swarming and twitching motility in a genome-wide deletion collection of P. aeruginosa strain PA14 (PA14 library). The protocol offers great versatility and ease of use, allowing modifications to different steps of the protocol, such as using different culture media or mutant collections.
The mutants were scored ...

This protocol presents a rapid and efficient method for identifying genetic factors involved in various types of motilities in Pseudomonas aeruginosa.

Motility behaviors often play a significant role in the ability of a bacterium to exploit the resources available in its environment. This is particularly ...

Our research focuses on understanding how bacteria work and cause infection using system biology and genomics. We aim to identify the genes that influence bacterial physiology in condition that mimic the infection environment in order to find new targets for developing better antibiotics and alternative treatments, We developed a high-throughput protocol to identify genetic factors influencing Pseudomonas aeruginosa motility, enabling genome-wide analysis of swarming and twitching behaviors. This research initiative uncovered molecular mechanism driving motility that also contribute to biofilm formation, colonization, and host defense evasion.It is adaptable to different assays and bacterial species. To begin, take the source plates of the Pseudomonas aeruginosa transposon mutant library and leave them flat on a surface at room temperature for approximately one hour to cool down. For the desired assay, select M9 glucose 0.5%agar plates for swarming and LB agar 1%agar plates for twitching.To configure the replicator for accurate colony transfer, turn on the replicator. In the section select and operate mode, choose select and run stored programs. In the section select source plates, select plus plate 384 agar.Then, in the select target plate section, select plus plate 384 agar. In the section select pads, select short pin 384. In singer programs, select replicate many, then go to replicate program options and choose general, recycle, and none.In the section source, select offset, then random and default radius. In the section target, select pinning and adjust the pressure to 2%Leave all other settings as default. Then, insert the short pin RePads 384 into the appropriate compartment.Position the source and target plates on the designated platform of the replicator. Place the 384 density source plates and the empty motility plates onto the platform. Press run using the selected parameters to begin the transfer process.The microbial array pinning robot will transfer colonies from the 384 density source plates to the motility plates. Afterward, place the inoculated motility plates into plastic bags. Carefully place the bags containing the plates into the incubator, set to 37 degrees Celsius for 18 hours.After the incubation period, capture high-resolution images of the motility plates using any high-quality imaging software. After performing the motility assay and imaging the motility plates, run the macro in ImageJ. Open the folder containing the images to load the batch for analysis.When prompted, modify the angle using the preview option until the plate is straight to adjust the plate's rotation. When satisfied, click the okay button. Then, move the rectangle tool around the colonies to crop the portion of the plate containing the colonies.When ready, click okay to complete the cropping. Define the grid used to draw the region of interest, or ROI, around each colony and measure the motility area. Select the appropriate colony density and adjust the parameters as needed to ensure that each circle is positioned around a colony.When ready, activate the okay box and click the okay button. The motility area for each colony will be measured, and a CSV file containing the data will be saved in the designated folder. For motility assay, autoclave an agar solution containing M9, glucose, casamino acids, and magnesium sulfate.Pour 25 milliliters of the cooled, mixed agar into each Petri plate. Then, inoculate each plate with 2.5 microliters of overnight bacterial culture and incubate at 37 degrees Celsius for 18 hours with the lids facing up to observe motility phenotypes. After autoclaving a solution of LB with 1%agar, pour 25 milliliters of the cooled, mixed LB 1%agar into Petri plates.Stab inoculate bacteria to the bottom of the twitching plates containing 1%agar. Incubate the plates at 37 degrees Celsius for 48 hours, and then at room temperature for an additional 48 hours. After incubation, carefully remove the agar from the plates.Visualize the twitching zone by staining the Petri dish with 1%crystal violet solution. The twitching motility assay showed that Pseudomonas aeruginosa mutants with T4P machinery gene deletions exhibited significantly smaller halo regions compared to the wild type, indicating reduced twitching motility. Swarming motility analysis revealed that deletion mutants of the PA14 library had significantly reduced halo regions with no protrusions, confirming defective swarming ability.Bacterial motility studies are often limited by their throughput. Our high-throughput motility protocol will help researchers study bacterial motility comprehensively. For instance, by using a genome-wide mutant collection, it becomes possible to identify all genes linked to motility and reveal how bacteria cause infections.Our findings raise new questions about the genetic control of motility in Pseudomonas aeruginosa, its role in biofilm formation, and its connection to pathogenesis. They also help with exploration of how environmental conditions shape motility behaviors, and whether targeting motility genes could lead to innovative antimicrobial therapies.

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Research

JoVE Journal

Immunology and Infection

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Source: Morales-Soto, N. et. al., Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays. J. Vis. Exp. (2015)This video demonstrates a swarming assay using bacteria that express green fluorescent protein to observe surface motility on soft agar. Time-lapse imaging captures coordinated expansion and tendril formation, highlighting bacterial surface motility.

Swarming Assay for Visualizing Bacterial Surface Motility

Source: Chu, W., and Zhuang, X. Visualizing Bacterial Motility Based on a Color Reaction. J. Vis. Exp. (2022)This video demonstrates a redox dye-based semisolid agar assay for assessing bacterial motility by visually distinguishing motile and non-motile strains.

Visualization of Bacterial Motility Using Redox Dye–Supplemented Semisolid Medium

Source: Scheidweiler, D., et al. Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales. J. Vis. Exp. (2020).This video demonstrates the use of a microfluidic device to quantify bacterial motility through a porous matrix composed of randomly arranged pillars. Fluorescently labeled bacteria are introduced into the device, imaged via microscopy as they move through the porous matrix into the observation channel, and analyzed...

Assessing Bacterial Motility Using a Microfluidic Device

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce's disease in grapevine.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

Visualizing Bacterial Motility Based on a Color Reaction

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize phenotypic variants, such as persisters, by time-lapse epifluorescence microscopy.

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Learn a high-throughput method for observing motility phenotypes in Pseudomonas aeruginosa using transposon mutant libraries and image analysis. This protocol enables genome-wide identification of genetic factors controlling swarming and twitching behaviors.

High-throughput Method for Observing Motility Phenotypes in Pseudomonas aeruginosa

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