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,....

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 ....

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