High-throughput Method for Observing Motility Phenotypes in Pseudomonas aeruginosa

Summary

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

Abstract

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.

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen that can colonize diverse environments because of its remarkable metabolic versatility^1,2. 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 body^3,4,5. 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 twitching^1,6. Each type of motility is mediated by distinct mechanisms and cellular structures and allows P. aeruginosa to respond to environmental cues^7,8. 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 surfaces^10,11. Finally, twitching motility is flagella independent. Instead, twitching is mediated by type IV pili (T4P) and used to move on top of surfaces^12,13. 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 biofilms^1,4,13.

Traditionally, bacterial motility assays are carried out individually, one strain at a time, on a soft agar plate or using microscopy methods^6,14,15. 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 µL 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 expansion^1,17,18.

More recently, transposon mutagenesis approaches have discovered new genes involved in motility or biofilm formation in P. aeruginosa^19,20,21. 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 collections^22,23. 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.

Protocol

NOTE: The general workflow of this procedure is outlined in 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. Please click here to view a larger version of this figure.

Swarming plates - M9 media	Twitching plates - LB media	Source plates - LB media
200 mL of a 5x M9 salts solution	10 g NaCl	10 g NaCl
10 mL of 20% glucose	10 g Tryptone	10 g Tryptone
25 mL of casamino acids	5 g Yeast Extract	5 g Yeast Extract
1 mL of 1M MgSO4	10 g Agar	15 g Agar
500 mL of 1% agar	Add dH2O to 1L	Add dH2O to 1L
Add dH2O to 1L		Gentamicin (final concentration 15 µg/mL)

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.

1. 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.
   1. 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.
   2. 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.
   3. Swarming plates: Prepare 15 (384-density format) or 59 (96-density format) swarming plates (M9-glucose 0.5% agar)
2. 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.
3. 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.

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.

1. Day 1: Replication of the PA14 library from the 59 frozen 96-well plates onto source plates
   1. 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.
   2. 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.
   3. 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.
2. Day 2: Upscaling from 96-density format to 384-density format
   NOTE: Day 2 can be skipped if working at 96-density.
   1. 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.
   2. 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.
   3. 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.
   4. In the section Replicate program options, select General and click on Recycle > None.
   5. In the Source section, select Offset and click on Random > Default Radius. In the section Target, select Pinning and adjust the pressure to 20%. Leave all other settings as default.
   6. Insert the Short Pin RePads 96 into the appropriate compartment.
   7. 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.
   8. 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.

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. Please click here to view a larger version of this figure.

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.

1. 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.
2. 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.
3. Configure the replicator with the following parameters to ensure accurate colony transfer.
   1. 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.
   2. 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 > Recycle > None.
   3. In Singer Programs, select Replicate Many.
   4. In the section Source, select Offset > Random > Default Radius. In the section Target, select Pinning and adjust the pressure to 2%. Leave all other settings as default.
4. Insert the Short Pin RePads 384 in the appropriate compartment.
5. 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.
6. 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.
7. After incubation, check the motility plates for even colony growth and absence of contamination.
8. 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.
   1. 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.
   2. A Select Project page opens; click on New Project > 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.
   3. 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.
   4. 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.
   5. 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.

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).

1. 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.
   1. Run the macro in ImageJ and open the folder containing the images.
   2. 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.
   3. 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.
   4. 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.
2. 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.

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.

1. Swarming motility assay
   NOTE: Swarming motility is performed as described in detail by Ha et al.¹⁶.
   1. 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).
   2. 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.
2. Twitching motility assay
   NOTE: Twitching motility is performed as described in detail by Haley et al.^33,34.
   1. Briefly, autoclave a LB 1% agar (see Table 1). Pour the cooled, mixed LB 1% agar into Petri plates (25 mL per plate).
   2. 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.
   3. Photograph the Petri dishes using the PhenoBooth with the same parameters described in step 3.

Results

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 ...

Discussion

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

Materials

List of materials used in this article

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