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

Summary

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.

Abstract

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The “border-crossing” assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Introduction

Bacteria employ many appendages for movement and dispersal in their ecological niches¹. Flagella-driven motility is the fastest of these, promoting the colonization of favorable locales in response to environmental signals, and contributing significantly to the pathogenic ability of some species^2,3. Flagellated bacteria can swim individually in bulk liquid, or swarm as a collective over a semi-solid surface⁴. Extracellular flagella attach to and are driven by rotary motors embedded in the membrane, which harness the power of ion gradients to generate torque that ....

Protocol

1. Isolation of suppressor mutants in motility-deficient strains

NOTE: Use this method as a broad ‘catch-all’ to identify the general nature of the motility defect.

1. Soft-agar plate preparation
   NOTE: Soft-agar, also referred to as motility- or swim-agar, is a low percentage agar (~0.2-0.35% w/v), long used to assay chemotaxis^31,32.
   1. Add 3 g of bacto-agar (0.3% w/v) and 20 g of LB to a 2 L round bottom flask. Add 1 L of ddH₂O (double-distilled water) to the flask and evenly mix the suspension using a stir rod and ma....

Results

The isolation of pseudo-revertants in an E. coli strain whose motility is impaired by high levels of the signaling molecule c-di-GMP, was detailed in recent work from our lab³⁴. This strain (JP1442) harbored two mutations: ΔyhjH and ΔycgR. YhjH is the most active phosphodiesterase that degrades c-di-GMP in E. coli. Absence of YhjH leads to elevated c-di-GMP levels and inhibition of motility. YcgR is a c-di-GMP effector. In complex with c-di-GMP, YcgR binds to th.......

Discussion

The isolation and characterization of suppressor mutations have successfully contributed to identifying key components of the chemotaxis system^35,36,37, as well as the motor machinery itself^38,39,40. While using Protocol 1, it is important to include multiple independent replicates to ensure the isolation of a large spectrum of possible.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Bacto Dehydrated Agar	Fisher Scientific	DF0140-15-4
EDTA Disodium Salt, Dihydrate	Fisher Scientific	02-002-786
Eiken agar	Eiken Chemical Co. Japan	E-MJ00	Essential for E. coli swarming
Glucose D (+)	Fisher Scientific	410955000
LB (Lennox) Broth	Fisher Scientific	BP1427-500
Poly-L-lysine Solution (0.1%)	Sigma-Aldrich	P8920
Potassium chloride (KCl)	Fisher Scientific	18-605-496
Potassium Phosphate monobasic (KH2PO4)	Fisher Scientific	BP362-500
Potassium Phosphate dibasic (K2HPO4)	Fisher Scientific	BP363-500
Sodium chloride (NaCl)	Fisher Scientific	S271-500
Materials and Equipment
CellSense microscope imaging software (V. 1.6)	Olympus		Or equivalent software for microscope used
Electron Microscopy Sciences Scotch 666 Doube Sided Tape	Fisher	50-285-28
Frosted microscope slides 3x1x1mm	Fisher	12-550-343
Olympus BX53 microscope	Olympus	BX53	Any upright or inverted phase microscope can be used
Petri dishes (100 mm diameter)	Fisher Scientific	FB0875712	For soft-agar assays
Polyethylene Nebulizer Capillary Tubing (0.58mm x 99mm 3.0m)	Perkin Elmer	9908265
Round Petri Dish with 2 Compartments	VWR	89200-944	For border-crossing assays
Safety Hypodermic Needles (23G)	Fisher Scientific	14-826A
Sterile Syringe - 1 mL	Fisher scientific	14-955-450
Task/Tissue wipes	Fisher scientific	06-666	Or equivalent single use tissue wipes
VWR micro cover-glass 18x18mm	VWR	48366205
XM10 camera	Olympus	XM10	Or equivalent microscope camera