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Method Article

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

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DOI:

10.3791/61364

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May 10th, 2020

 , 
1Department of Molecular Biosciences, The University of Texas at Austin

In This Article

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 species2,3. 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 causes rotation1,2,4,5,6,7,8. In E. coli, whose motors run at a constant torque9, the motor output can be categorized in terms of rotational speed and switching of the rotor between counter-clockwise (CCW) and clockwise (CW) directions. CCW rotation promotes formation of a coherent flagellar bundle that propels the cell forward (run), while a transient switch in rotational direction (CW) causes the bundle to disassemble either partially or fully10, and the cell to reorient its swimming direction (tumble). E. coli typically run for a second and tumble for a tenth of a second. Switching frequency of the rotor or ‘tumble bias’ is controlled by the chemotaxis signaling system, wherein transmembrane chemoreceptors detect external chemical signals and transmit them via phosphorelay to the flagellar motor to extend runs in response to attractants, or suppress them in response to toxic chemicals11,12. Swimming motility is assayed in 0.3% soft agar.

During swarming, bacteria navigate on a semi-solid surface as a dense collective, where packs of bacteria stream in a continuous swirling motion2,13,14,15. E. coli swarms exhibit altered chemosensory physiology (lower tumble bias), higher speeds, and higher tolerance to antimicrobials over cells swimming in bulk liquid16,17. Swarmers vary in their deployment of a plethora of strategies that aid movement, including surfactant production, hyperflagellation, and cell elongation2. Swarming offers bacteria a competitive advantage in both ecological and clinical settings18,19,20. There are two categories of swarming bacteria: temperate swarmers, which can swarm only on media solidified with 0.5-0.8% agar, and robust swarmers, which can navigate across higher agar concentrations21.

A variety of assays exist to interrogate swimming motility and its regulation. When impaired by mutations or environmental conditions, motility itself offers a strong selection for identifying gain-of-function suppressor mutations. These suppressors can be genuine revertants of the original mutation, or pseudo-revertants, where a second mutation restores functionality. Such mutants can be identified by whole genome sequencing (WGS). An alternative to unbiased suppressor selection is a biased targeted mutagenesis strategy (e.g., PCR mutagenesis). These methodologies often shed light on the function or environmental regulation of the motility apparatus. If the goal is to study motor function, then the restoration of wild-type motility as measured in soft agar may not necessarily indicate restoration of wild-type motor output. The cell-tethering assay, in which cells are attached to a glass surface by a single flagellum and rotation of the cell body is subsequently monitored, can be the initial assay of choice for assessing motor behavior. Although more sophisticated methodologies are now available to monitor motor properties, the required high-speed camera set-up and application of software packages for motion analysis limit their widespread use22,23,24,25. The cell-tethering assay requires only that the flagella be sheared, allowing attachment of the short filaments to a glass slide, followed by videotaping the rotation of the cell body. Although the recorded motor speeds are low in this assay because of the high load the cell body exerts on the flagellum, this assay has nonetheless contributed to valuable insights into chemotactic responses26,27,28,29, and remains a valid investigative tool as discussed below.

Swarming motility poses a different set of challenges to researchers. Selection of gain-of-function suppressors only works in swarmers that produce copious surfactants and swarm readily13. Surfactant non-producers such as E. coli are fastidious with respect to the choice of agar, media composition and humidity of the environment2,13,14,21. Once swarming conditions are established, the border-crossing assay17 is a useful methodology to interrogate the ability of a swarm to navigate new/harsh conditions. Though the protocols presented below relate to E. coli, they can be readily adapted for application in other species.

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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 chemotaxis31,32.
    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 magnetic stirring plate.
    2. Autoclave for 20 min at 121 °C.
    3. Allow to cool with gentle agitation using the rod/plate as above. When the temperature reaches approximately 50 °C, pour 25 mL into sterile Petri dishes (100 mm x 15 mm), and allow the molten agar to set with lid in place for at least 1 h, for use within 16 h.
  2. Culture preparation, inoculation, and isolation of suppressor mutants
    NOTE: E. coli inoculated in the center of nutrient rich media solidified with soft agar consume nutrients locally, creating a nutrient gradient that they follow. As they move outward, defined ‘rings’ appear (Figure 1A), which are related to specific chemoattractants the bacteria respond to. Defects in either the chemotaxis system or structural components of the flagella motor can compromise performance in this assay. Often, mutants with a motility advantage arise during screening, and can be seen emerging from single or from multiple points along the periphery of the ring, from where they ‘flare’ out (Figure 1C). One will notice that the outermost edge of the swimming front contrasts readily against the uncolonized virgin soft agar.
    1. Grow overnight cultures of the desired motility-deficient strain in 5 mL of Lennox Broth (LB; 10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl, Table of Materials) at 30 °C with horizontal shaking (220 r.p.m.). The following day, sub-culture (1:100 dilution) in fresh LB, growing under the same conditions to exponential phase (OD600 of 0.6).
    2. Inoculate 6 µL of the culture into the center of a soft-agar plate (1.1) using a pipette, pushing the loaded sterile tip into the agar to gently expel the contents. Transfer to 30 °C and incubate (Figure 1B), until motility ‘flares’ are evident, emanating from the inoculation point or the periphery of the motility rings, typically in 24-36 h (Figure 1C).
      NOTE: In motility assays, inoculate a wild-type strain alongside the mutant isolates for comparison. This wild-type strain will show the characteristic concentric chemotactic rings (Figure 1A) and fill the plate within 8-10 h.
    3. Use a sterile wire-loop to lift the cells from the ‘flare’ region and streak to purify single colonies onto an LB hard agar plate (LB prepared as above, solidified with 15 g/L bacto-agar).
    4. Pick single colonies from the streak plate using a sterile wire loop and re-purify by streaking for single colonies to ensure isolation of a ‘pure’ colony isolate.
  3. Confirmation, and characterization of suppressor mutants
    1. Confirm that the isolated suppressor mutant(s) have restored motility. Prepare soft-agar plates (1.1), and cultures for strains of interest (as in 1.2.1), including wild-type and the starting ‘motility-deficient’ strain for comparison.
    2. Inoculate the plates (as in 1.2.2) and incubate at 30 °C for 8-10 h.
    3. Record the diameter of the outermost ring (edge of the circle) and compare to establish which of the isolates have substantially restored motility.
      NOTE: It is recommended that plates be photographed throughout the time-course of the experiment. For best results, use a “bucket of light” device30, where a digital camera is mounted above a light source for better illumination to measure the diameter of the swim colony and distinguish it from uncolonized agar.
    4. Subject verified isolates to WGS as required, allowing for sufficient ‘sequence coverage’ to positively identify the mutations that restored wild-type function.

2. Quantifying flagella motor behavior via cell tethering

NOTE: Use this method when normal run-tumble behavior (chemotaxis) appears to be compromised.

  1. Culture preparation and flagella shearing
    1. Prepare an exponential phase culture of the strain of interest as described in step 1.2.1.
    2. Pellet 10 mL of cells by centrifugation at 2,000 x g for 3 min before resuspending in 10 mL of filter-sterilized Motility Buffer (MB; 10 mM potassium phosphate buffer [0.0935 M K2HPO4, 0.0065 M KH2PO4, pH 7.0], 0.1 mM EDTA [pH 7.0], 10 mM NaCl, 75 mM KCl).
      NOTE: MB supports motility, but does not support bacterial growth
    3. Repeat step 2.1.2 two more times before resuspending the final pellet in 1 mL of MB.
    4. Transfer the cell suspension into a 1 mL syringe and attach a 23G needle to the end. Assemble an identical syringe/needle apparatus and attach the two together via 6 inches of polyethylene tubing (inner diameter of 0.58 mm) tightly sheathed over each needle tip.
    5. Shear the flagella (they are fragile and break easily) by gently passing the cell suspension back and forth from one syringe to the other 50x, with 1 min pauses between every 10 passes.
    6. Centrifuge the sheared cells at 2,000 x g for 3 min and resuspend in a final volume of 500 µL of MB.
  2. Slide preparation and cell tethering
    1. Prepare a cell fixation chamber by stacking an 18 mm x 18 mm coverslip over a 3 inch x 1 inch x 1 mm glass microscope slide, separated by double-sided tape (Figure S1).
    2. Flush the chamber with 0.01% (w/v) poly-lysine solution by applying to the top of the chamber. Tilt the bottom edge (the coverslip flush with the microscope slide) onto a task wiper (tissue paper) to help draw solution through the chamber (Figure S1). Then incubate at room temperature for 10 min.
    3. Wash the chamber three times with 40 µL of MB using as described in step 2.2.2
    4. Add 40 µL of the sheared cell suspension (prepared above) to the top of the chamber and incubate at room temperature for 10 min to allow cells to attach to the coverslip.
    5. Gently flush the chamber with 40 µL of MB as in 2.2.3 to remove unattached cells.
  3. Cell rotation recording and quantification
    1. Transfer the microscope slide loaded with tethered cells to the microscope stage.
    2. Using phase-contrast microscopy and a 100x objective, scan the population for cells that are fixed in place, and rotating on a single axis, i.e., smooth rotations on a fixed point rather than presenting at an angle wherein the cell moves in and out of focus (Video 1).
    3. Use a commercial microscope and associated camera. Open the associated software, ensure cells of interest are in focus and click video acquisition to record the cell rotation for one minute (at 10 frames per second or higher).
    4. From video playback, quantify the number of complete rotations per minute and the number of times the cell changes direction (switching frequency).
      NOTE: Rotational speeds and switching frequency may be too fast to gauge by eye so it is recommended to use video software that offers slow/fine playback, or adopts an automated software system to quantify rotational patterns33. An alternative would be to increase the viscosity of the MB using methyl cellulose (or similar agent) to help slow and resolve the rotation of faster bacteria or compensate when cameras with low framerates are in use.
    5. Repeat step 2.3.2 with biological replicates to compile a representation of the population of interest.

3. Preparation of swarms in a border-crossing assay

NOTE: Use this method for assessing the impact of a mutation or condition on group motility. Swarm-agar refers to agar where the percentage is typically higher than that of soft-agar. In soft agar (0.3 %), cells swim individually inside the agar. In swarm agar (0.5% and above), cells move as a group on the surface. While swarm plates must be used as detailed here, swim plates have a longer shelf life, and may be used for several days. Our personal preference is to use in 1-2 days.

  1. Preparation of swarm agar
    1. Add 5 g of Eiken agar (0.5% w/v) and 20 g of LB to a 2 L round bottom flask. Add 1 L of ddH2O to the flask and evenly mix the suspension using a stir rod and magnetic stirring plate.
    2. Autoclave for 20 min at 121 °C.
    3. Allow to cool with gentle agitation to avoid any air bubbles using the rod/plate as above. When approximately 50 °C, add filter-sterilized glucose for a final concentration of 0.5 %.
    4. Pour 25 mL into sterile Petri dishes (100 mm x 15 mm) and allow to set at room temperature for at least 14 h and no more than 20 h. Do not store for future use.
  2. Inoculation and incubation of swarm plates
    1. Inoculate 6 µL of a mid-exponential culture (prepared as in 1.2) by spotting on top of the agar.
    2. Leave the lid off for 5-10 min and replace when the inoculum has dried into the agar surface.
    3. Incubate at 30 °C for 8 h. Avoid the temptation to inspect swarm progress by removing the lid, as this will contribute to drying of the agar and impair swarming.
      NOTE: Incubation time may vary depending on the strain phenotype. Some isolated mutations may hamper swarming ability and will require a reduced percentage of agar or a more prolonged period of incubation.
  3. Preparation of border-crossing assay plates
    NOTE: This assay utilizes a modified Petri dish, where a plastic divide (border) creates two chambers, rather than one (Figure 2A). Each chamber can be prepared independently of the other, offering differing conditions for swarming, prior to ‘connecting’ the two. Depending on experimental design, the first chamber (designated left) can be prepared with either swim agar (0.3% w/v) or swarm agar (0.5% w/v) from where the bacteria can migrate across the border into the right chamber containing swarm agar +/- any required supplement or challenge (e.g., antibiotics). Migration on either agar is typically measured/compared by recording the widest diameter of bacterial colonization (edge to edge) from the original point of inoculation.
    1. Prepare swim agar as described in 1.1 if required.
    2. Prepare swarm agar as described in 3.1.
    3. Pour ~30 mL of swarm agar (with desired supplementation if required) into the right chamber of a dual-compartment Petri dish (100 mm x 15 mm), to the point where it is level with the plastic divider between chambers, but not overflowing into the left (Figure 2B).
    4. After the agar has hardened, fill the left chamber with ~30 mL of swim or swarm agar, again to the point of contact with the plastic divide (Figure 2C). Before it sets, use a sterile pipette tip to gently drag the agar over the border to connect the two sides with a ~1 mm tall agar bridge that spans the entire length of the divide (Figure 2D).
    5. Allow the plate to dry at room temperature (3.1).
      NOTE: An alternative method of creating bridge is allow the left chamber agar to dry and then slowly pipette ~100 μL of molten swarm agar along the plastic divider to bridge the two chambers (3.4). Inoculate the plates on the left chamber as detailed above for swim (1.2.2) or swarm (3.2) agar, before incubating at 30 °C for 12-16 h, or until swarms have made sufficient progress over the right chamber to allow comparisons between strains of interest.

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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...

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Discussion

The isolation and characterization of suppressor mutations have successfully contributed to identifying key components of the chemotaxis system35,36,37, as well as the motor machinery itself38,39,40. While using Protocol 1, it is important to include multiple independent replicates to ensure the isolation of a large spectrum of possible...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by National Institutes of Health grant GM118085 and in part by the Robert Welch Foundation (grant F-1811 to R.M.H.).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Reagents
Bacto Dehydrated AgarFisher ScientificDF0140-15-4
EDTA Disodium Salt, DihydrateFisher Scientific02-002-786
Eiken agarEiken Chemical Co. JapanE-MJ00Essential for E. coli swarming
Glucose D (+)Fisher Scientific410955000
LB (Lennox) BrothFisher ScientificBP1427-500
Poly-L-lysine Solution (0.1%)Sigma-AldrichP8920
Potassium chloride (KCl)Fisher Scientific18-605-496
Potassium Phosphate monobasic (KH2PO4)Fisher ScientificBP362-500
Potassium Phosphate dibasic (K2HPO4)Fisher ScientificBP363-500
Sodium chloride (NaCl)Fisher ScientificS271-500
Materials and Equipment
CellSense microscope imaging software (V. 1.6)OlympusOr equivalent software for microscope used
Electron Microscopy Sciences Scotch 666 Doube Sided TapeFisher50-285-28
Frosted microscope slides 3x1x1mmFisher12-550-343
Olympus BX53 microscopeOlympusBX53Any upright or inverted phase microscope can be used
Petri dishes (100 mm diameter)Fisher ScientificFB0875712For soft-agar assays
Polyethylene Nebulizer Capillary Tubing (0.58mm x 99mm 3.0m)Perkin Elmer9908265
Round Petri Dish with 2 CompartmentsVWR89200-944For border-crossing assays
Safety Hypodermic Needles (23G)Fisher Scientific14-826A
Sterile Syringe - 1 mLFisher scientific14-955-450
Task/Tissue wipesFisher scientific06-666Or equivalent single use tissue wipes
VWR micro cover-glass 18x18mmVWR48366205
XM10 cameraOlympusXM10Or equivalent microscope camera

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