High-throughput Method for Observing Motility Phenotypes in Pseudomonas aeruginosa

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.