Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Summary

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

Abstract

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Introduction

Bacteria constantly experience changes to their external environment and have developed several techniques to change and adapt their behavior accordingly. One such mechanism involves differentiation into distinct cell types that better complement the altered environment. Differentiation often involves major changes in the regulation of the cell cycle, cell morphology, and the spatiotemporal organization of the cells. One organism that can undergo differentiation is Vibrio parahaemolyticus. V. parahaemolyticus belongs to the Vibrionaceae, whichis a family of proteobacteria that usually inhabit fresh or salt water. Vibrionaceae are widely distributed in the environment and include several species that cause intestinal tract infections in humans, also including Vibrio cholerae.V. parahaemolyticus is a dimorphic organism and is able to differentiate into two distinct cell types as a response to accommodate changes to its external milieu. In aqueous environments, it exists as a short rod-shaped swimmer cell with a single polar flagellum positioned at the old cell pole. Upon surface contact, differentiation into a swarmer cell is triggered. Swarmer cell differentiation involves two major changes: swarmer cell morphogenesis through inhibition of cell division, and the induction of a second flagella system. This results in the formation of a peritrichous and highly elongated rod-shaped swarmer cell, which can either continue the swarmer life-style, where division events results in progeny swarmer cells, or alternatively differentiate back into swimmer cells.

Several factors have been reported to induce or influence swarmer differentiation. The primary stimulus appears to be driven by mechanosensing where V. parahaemolyticus uses the polar flagellum as a tactile sensor that detects inhibition of the rotation upon surface contact, but several other factors are involved as well¹. In the lab, rotation of the polar flagellum can be artificially inhibited by addition of phenamil, which blocks the sodium-channel driven flagellar rotation, thereby inducing swarmer differentiation². Furthermore, in an earlier study, Vibrio alginolyticus was induced to swarm on solid media when cells were propagated on growth medium in a Petri dish sealed with clear plastic tape. Alkali-saturated filter paper prevented swarming under these conditions, hence suggesting that one or more volatile acids might be involved in induction of swarming. Thus, sealing the Petri dish with plastic tape likely allowed for the accumulation of volatile acids, formed as by-products of cellular metabolism, within the head space of the plate. The same effect was achieved when H₂O₂ was added to the growth medium in un-sealed Petri dishes in order to artificially produce volatile acids by hydrolysing media components^3,4,5. Nevertheless, the identity of such volatile acids remains unknown. Moreover, it has been shown that excess availability of calcium⁶ and iron-limitation⁷ both enhance swarmer differentiation and proliferation over solid surfaces. Cells can be starved for iron by adding the compound 2,2´-Bipyridyl to the growth medium, which has been shown to influence swarmer differentiation⁸. The factors known to regulate differentiation are now implemented in the design of a protocol that reproducibly induces V. parahaemolyticus differentiation into swarmer cells and their proliferation on solid agar surfaces.

V. parahaemolyticus differentiation involves major changes in the regulation of cell division, cellular morphology, and the positioning of macromolecular machines such as flagella and chemotaxis apparatuses – processes that all require the specific localization of proteins in accordance with the cell cycle. Thus, the ability to study the intracellular localization of such proteins is essential to the understanding of the aforementioned cellular processes. In order to perform such studies fluorescence microscopy on single cells is required. This can be particularly challenging when imaging cells that exist within dense bacterial populations, as is the case for swarmer cells. There have been attempts to induce swarmer differentiation in liquid media, which could potentially generate single cells for microscopy studies. And although on a transcriptional level these cells have partially induced the swarmer-differentiation program, they do not undergo the same distinct morphological changes as fully differentiated swarmer cells grown on solid medium⁸. This paper offers a robust and reproducible protocol of a method to induce swarmer cell differentiation on an agar surface. The protocol produces a population of easily accessible swarmer cells readily available for single cell microscopy and subsequent analysis. Furthermore, the protocol enables localization studies of fluorescently labeled proteins in both the swimmer and swarmer cell-types (sections 1, 2, 3, 4). Additionally, the protocol describes the subsequent workflow on how to process and analyze the generated data from fluorescence microscopy experiments, which allows for demographic analysis of bacterial societies (section 5).

Protocol

1. Preparation of Electro-competent V. parahaemolyticus Cells and Electroporation of Plasmid DNA into Swimmer Cells

NOTE: The following section of the protocol allows for the preparation of electro-competent cells and the subsequent electroporation of plasmid DNA into swimmer cells. This step is important if fluorescent proteins are being ectopically expressed from a plasmid.

1. Streak out V. parahaemolyticus cells from a -80 °C glycerol stock onto a fresh LB agar plate containing 100 µg/mL ampicillin and incubate overnight at 37 °C.
2. The following day, inoculate 200 mL of LB medium with a single colony of V. parahaemolyticus and incubate it at 37 °C under shaking conditions until an OD₆₀₀ of 1.0 is reached. It usually takes 5-6 h of incubation for the cell culture to reach the desired optical density.
3. Immediately transfer the cells onto ice and perform all further steps on ice and in 4 °C pre-cooled centrifuges.
4. Harvest the cells at 4 °C for 10 min at 2,000 x g.
5. Discard the supernatant by decantation and re-suspend the cell pellet in 25 mL of ice-cold 273 mM sucrose solution (pH 7.4, buffered with KOH).
6. Harvest the cells again at 4 °C for 10 min at 2000 x g. Repeat two additional times.
7. Re-suspend the washed cell pellet in 400 µL of ice-cold 273 mM sucrose solution. Subsequently, add glycerol to a final concentration of 15% vol/vol. The cells are now ready for electroporation, but can also be frozen in aliquots of 70 µL at -80 °C for later use.
8. For the electroporation, mix a 70 µL aliquot of electro-competent cells with 100 - 1,000 ng of plasmid DNA and transfer the mixture into an ice-cold electroporation cuvette (0.2 cm electrode gap). Perform electroporation with the following settings: 25 µF, 2400 V and 200 Ω.
9. Suspend the cells in the electroporation cuvette in 600 µL of LB broth, transfer to a 2 mL tube and incubate while shaking at 37 °C for 3 h.
10. Spin down the cells at 2,000 x g for 5 min and re-suspend the pellet in 100 µL of LB broth. Spread the cell-suspension onto selective LB agar plates containing the necessary antibiotics and incubate at 37 °C overnight.
11. The following day, check the plates for colony formation. Colonies that grow carry the correct antibiotic resistance gene encoded on the plasmid and can now be used for further experiments.

2. Induction of Swarmer Cell Differentiation

NOTE: The following steps describe how to induce differentiation of V. parahaemolyticus into its swarmer cell life-cycle and how to stimulate proliferation on solid surfaces.

1. Suspend 4 g of Heart Infusion (HI) Agar in 100 mL ddH₂O in order to prepare the swarming plates.
2. Carefully boil the HI agar solution in the microwave and shake the bottle from time-to-time until the powder is dissolved. Autoclave at 121 °C for 20 min.
3. After autoclaving, cool down the HI agar to 60-65 °C. Add 2,2´-Bipyridyl to a final concentration of 50 µM (stock solution of 50 mM in 100% ethanol). Add CaCl₂ to a final concentration of 4 mM (stock solution of 4 M in ddH₂O). If necessary, add antibiotics into the solution to maintain plasmids.
   NOTE: If planning to use an inducible plasmid, add inducing agent to its final concentration.
   CAUTION: 2,2´-Bipyridyl: Acute toxicity (oral, dermal, inhalation). Use gloves and safety goggles when handling this chemical.
4. Pour 30 - 35 mL of the final HI agar solution into a round 150 mm Petri dish and let the agar solidify.
5. Right before spotting the cell culture, dry the agar plate at 37 °C for at least 10 min (or until any liquid residues have disappeared, but no longer) up-right with an open lid.
   NOTE: Drying times highly depend on the incubator used and heavily dried plates will not permit swarming of V. parahaemolyticus. (This step is very important!) Plates must be used fresh and should not be older than 2 - 3 h. Old plates will be too dry to stimulate swarming.
6. Inoculate a small amount of cells from the edge of a single colony into 5 mL of LB broth. Incubate the culture shaking at 37 °C until OD₆₀₀ = 0.8 is reached, it usually takes around 2-3 hours. After that, the cells are ready to be spotted onto HI swarming agar plates.
7. Spot 1 µL of cell culture at the center of a HI agar swarming plate (Figure 1A).
8. Let the spot dry (Figure 1B).
9. Seal the plate with clear plastic tape and make sure that it is firmly attached, otherwise it might detach during overnight incubation (Figure 1C).
   NOTE: It is very important that the seal is perfect. Avoid the formation of air-bubbles and folds to the tape when it is applied.
10. Incubate the plate at 24 °C overnight. This will result in the formation of a V. parahaemolyticus swarm-colony with swarm-flares extending from the periphery of the swarm-colony (Figure 2).

3. Preparation of V. parahaemolyticus Swarmer Cells for Fluorescence Microscopy

NOTE: The following protocol describes how to prepare microscopy agarose slides and the subsequent imprinting of swarming flares onto an agarose pad to obtain single swarmer cells for microscopy.

1. To prepare agarose slides for microscopy, add 1 g of agarose to 100 mL of a 20% vol/vol PBS and 10% vol/vol LB broth solution. Carefully boil the solution in the microwave to dissolve the agarose and let it cool down to 65-70 °C while mixing with a magnetic stirrer.
2. Place a clean microscope slide onto a perfectly level section of a working bench. Cover both ends of the slide with two layers of general-purpose laboratory labeling tape, fixing the slide to the working bench. The distance between the two pieces of tape should be around 3 cm.
   NOTE: The two layers of tape create a very small elevation, which will determine the height of the agarose pad. It is important that the working space is level to make sure cells will be in the same plane for microscopy analysis.
3. Pipet ~ 250 µL of the previously prepared agarose solution onto the non-covered glass surface.
4. Now place a second microscope slide in the same orientation as the bottom one on-top and slightly press it down, so that residual agarose can flow out to the sides.
5. Let the agarose solidify 1-2 min and then slowly pull the top glass slide off the bottom one in a horizontal movement.
6. Using a scalpel, cut off any residual agarose that is attached to the sides of the microscope slide and slowly remove the tape from the ends of the microscope slide.
   NOTE: The agarose slide is now ready and should be used as soon as possible as it will slowly begin to dry out.
7. To perform microscopy on swarmer cells, cut out a ~3 mm x 10 mm piece of swarming agar from the most outer edge of a swarming colony (Figure 1D). It is very important not to break the tape seal of the swarm-plate until immediately before transfer of swarmer cells to the microscope slide. Once the tape is removed, swarming V. parahaemolyticus cells initiate differentiation into swimmer cells.
   NOTE: Make sure to direct the cuts from the outside of the swarming colony edge to the inside of the colony (Figure 1D). This way you can prevent "swimmer contamination" into the edge of the swarming colony.
8. Transfer the piece of swarming agar onto a microscope agarose slide with the cells facing the agarose pad (Figure 1E). This imprints the swarming cells onto the agarose pad.
9. After waiting ~30 s, remove the agar piece carefully from the agarose pad (Figure 1F).
10. Place a glass coverslip onto the agarose pad where the cells were imprinted. The cells are now ready for microscopy.
    NOTE: It is important to image swarmer cells immediately, since swarmer cells slowly initiate differentiation to the swimmer state.

4. Preparation of V. parahaemolyticus Swimmer Cell-cultures For Fluorescence Microscopy

NOTE: This section of the protocol describes how to prepare a culture of V. parahaemolyticus to perform fluorescence microscopy on the swimmer cell-type.

1. Inoculate a small amount of cells from the edge of a single colony into 5 mL of LB broth. Incubate the culture shaking at 37 °C for 1 h or until slight cell-growth becomes visible.
2. At this stage, if needed, add inducing agent for expression of proteins of interest. In this example, L-arabinose was added to a final concentration of 0.2% (w/vol) in order to induce expression of YFP-CheW⁹.
3. Incubate the culture shaking for an additional 2 h at 37 °C.
4. Follow steps 3.1-3.7 to prepare microscopy agarose slides.
5. Spot 1 µL of cell culture at the center of the agarose pad and let the spot incubate until it is dry.
6. Place a glass coverslip onto the agarose pad where the cells were spotted. The cells are now ready to be imaged under the fluorescence microscope.

5. Image Analysis

This part of the protocol describes a workflow for image analysis, particularly how to extract fluorescence data of single cells for demographic analysis.

1. Before beginning the image analysis make sure that all microscopy images are saved in 16bit TIF format.
2. Load the DIC (or phase contrast) and the corresponding fluorescent channel images into the software.
3. Generate an overlay image of both channels by selecting "Display/Overlay images". Set the number of pictures ("# Images:") to two, and select the DIC image in the first channel and the fluorescent image in the second channel.
4. Select the "Multi-Line" tool from the toolbar and mark cells from one pole to the other through the middle of the cell. In order to get a list of all generated lines (line objects) open "Measure/Region Measurements". Configure the region measurements to only display the region label, distance, and average intensity.
5. Calibrate the pixel size under "Measure/Calibrate Distances" to the pixel size that is specific for your microscope setup and click "Apply To All Open Images".
6. Transfer all regions (line objects) to the fluorescent channel image. Under "Regions/Transfer Regions" select the source image (the overlay image) and the destination image (the fluorescent channel image). Select "All Regions" and press "OK".
   NOTE: To be able to later re-produce the exact lines (line objects, regions, ROI), open "Regions/Save Regions" and save the regions.
7. Export all region measurements into a spreadsheet by opening "Measure/Region Measurements" and pressing "Open Log". Confirm to open and export the data. Once the spreadsheet is opened in the background, finally export the data by pressing "F9: Log Data".
8. To determine the position of fluorescent foci along the cell, open "Measure/Linescan". Set "Linescan Width" to a value so that the whole width of a cell is covered by the region that you previously created. Right-click into the line-scan graph and select "Show Graph Data". By left-clicking and dragging the mouse-courser along the line-scan to the point of interest, the "Graph Data" window will display the corresponding pixel/distance between the two points. Select the blue highlighted row and then copy the content.
   NOTE: The entire line-scan intensity profile can be logged to a spreadsheet by pressing "Log Data" in the "Linescan" window. The extracted data can now be used for visualization in graphs or further statistical analysis. Localization patterns over the cell cycle can be easily visualized in a demographic representation of the cell length and the fluorescent intensities along the cell body. These representations can be obtained using the same ROI's previously selected. The following steps will give an example of how to generate demographics similar to the ones shown in Figures 3C and 4C.
   1. In Fiji/ImageJ load region markings (.rgn) previously created in the software (step 5.6) using the "Metamorph nd & ROI files importer". Alternatively, regions of interest (ROIs) of the cells to be analyzed can be created directly in Fiji/ImageJ using the build-in "Segmented Line" tool.
   2. Open "Analyze/Tools/ROI manager" and mark all active list entries".
   3. Afterward, click "More/Multi Plot" and then select "List" in the new "Multi Plot" window. Click on a table entry, then select and "copy all". Subsequently, paste into a new spreadsheet.
      NOTE: Make sure that the data of the longest cell (two corresponding X and Y columns with the most number of rows) is the last one to the right in the spreadsheet.
   4. Save the spreadsheet as a "CSV (Comma delimited) (*.csv)".
   5. Download the R scripts from here "https://github.com/ta-cameron/Cell-Profiles”¹⁰. The scripts sort cells by length and normalize the fluorescent intensity profiles of all cells as an average of each cell’s fluorescence.
   6. Run the entire script "cell profiles function.R" in R [version 3.0.1;¹¹].
   7. Edit the "example plots.R" script in line 30 such that the file path refers to the folder where the previously generated .csv file is saved. Additionally, change the file name "profile.csv" in line 31 to the file name chosen for .csv file generated in step 5.9.4. Afterwards, run "example plots.R" and follow the documentation from "https://github.com/ta-cameron/Cell-Profiles” as well as the comments from the script file to generate demographic analysis.

Results

Induction of differentiation and generation of swarming colonies

Figure 1 provides a schema of the important steps involved in producing swarming colonies of V. parahaemolyticus (Figure 1A-C). A swimmer culture was spotted on swarm-agar and incubated at 24 ^oC, inducing swarmer differentiation and proliferation over the solid agar surface. A representative stereo-microscopy image...

Discussion

This paper reports a method for microscopy imaging of V. parahaemolyticus swarmer cells, followed by downstream analyses intended to resolve and quantify the subcellular localization and other features of the studied fluorescently labeled proteins. Although several tools exist for microscopy image processing and analysis^{14,15,16,17,18}, the analysis of...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Media components
LB medium	Roth	X968.3
Difco Agar, granulated	BD	214510	For preparation of LB agar with LB medium
Difco Heart Infusion Agar	BD	244400	For preparation of HI agar swarming plates
Agarose NEEO, ultra quality	Roth	2267.3	For preparation of microscopy agarose pads
Name	Company	Catalog Number	Comments
Additives
L-arabinose	Roth	5118.3	Used to induce pBAD derivatives
2,2`-Bipyridyl	Sigma Aldrich	D216305-25G	For preparation of HI agar swarming plates
Calcium chloride dihydrate	Roth	5239.1	For preparation of HI agar swarming plates
Ampicillin sodium salt	Roth	K029.3
Chloramphenicol	Roth	3886.3
PBS buffer	-	-	Standard recipe for Phosphate-buffered saline
D(+) Sucrose	Roth	4621.1	For preparation of electrocompetent cells
Glycerol	Roth	3783.5	For preparation of electrocompetent cells
Name	Company	Catalog Number	Comments
Hardware
Petri dish 150 mm x 20 mm with cams	Sarstedt	82.1184.500	For preparation of HI agar swarming plates
kelvitron t (B 6420)	Heraeus	-	Used for drying HI agar swarming plates
Gene Pulser Xcell Microbial System	BioRad	1652662	Used for elctroporation of plasmid DNA
Name	Company	Catalog Number	Comments
Software
MetaMorph Offline (version 7.7.5.0)	Molecular Devices	-	Used for microscopy image analysis
Excel	Microsoft	-	Used for microscopy data analsis