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Özet

Presented here is a protocol that describes monitoring of the complete life cycle of predatory bacterium Bdellovibrio bacteriovorus using time-lapse fluorescence microscopy in combination with an agarose pad and cell-imaging dishes.

Abstract

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. Therefore, it is considered a living antibiotic. To apply B. bacteriovorus as a living antibiotic, it is first necessary to understand the major stages of its complex life cycle, particularly its proliferation inside prey. So far, it has been challenging to monitor successive stages of the predatory life cycle in real-time. Presented here is a comprehensive protocol for real-time imaging of the complete life cycle of B. bacteriovorus, especially during its growth inside the host. For this purpose, a system consisting of an agarose pad is used in combination with cell-imaging dishes, in which the predatory cells can move freely beneath the agarose pad while immobilized prey cells are able to form bdelloplasts. The application of a strain producing a fluorescently tagged β-subunit of DNA polymerase III further allows chromosome replication to be monitored during the reproduction phase of the B. bacteriovorus life cycle.

Introduction

Bdellovibrio bacteriovorus is a small (0.3–0.5 µm by 0.5–1.4 µm) gram-negative bacterium that preys on other gram-negative bacteria, including harmful pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and Shigella flexneri^1,2,3. Since B. bacteriovorus kills pathogens, it is considered a potential living antibiotic that can be applied to combat bacterial infections, particularly those caused by multidrug-resistant strains.

B. bacteriovorus exhibits a peculiar life cycle consisting of two phases: a free-living non-replicative attack phase and an intracellular reproductive phase (Figure 1). In the free-living phase, this highly motile bacterium, which moves at speeds of up to 160 µm/s, searches for its prey. After attaching to the prey’s outer membrane, it enters the periplasm^4,5. During the interperiplasmic reproductive phase, B. bacteriovorus uses a plethora of hydrolytic enzymes to degrade the host’s macromolecules and reuse them for its own growth. Soon after invading the periplasm, the host cell dies and bloats into a spherical structure called a bdelloplast, inside which the predatory cell elongates and replicates its chromosomes. The replication process starts at the replication origin (oriC)⁶ and proceeds until several copies of the chromosome have been completely synthesized⁷. Interestingly, replication of each chromosome is not followed by cell division. Instead, the predator elongates to form a long, multinucleoid and filamentous cell. Upon nutrient depletion, the filament undergoes synchronous septation and progeny cells are released from the bdelloplast⁸.

Before B. bacteriovorus can be used as a living antibiotic against bacterial infections, it is crucial to understand the major stages of its life cycle, particularly those related to its proliferation inside the prey. Live-cell imaging of B. bacteriovorus has been challenging, due to the various morphological forms of the predator and its prey during the complex life cycle. So far, the interactions between B. bacteriovorus and its host cell have been mainly studied by electron microscopy and snap-shot analysis^2,9,10, both of which have limitations, especially when they are used to monitor successive stages of the predatory life cycle. These methods provide high-resolution images of B. bacteriovorus cells and enable observation of a small predator during the attack or growth phase. However, they do not allow tracking of single B. bacteriovorus cells throughout both life cycle phases.

Presented here is a comprehensive protocol for using time-lapse fluorescence microscopy (TLFM) to monitor the complete life cycle of B. bacteriovorus. A system consisting of an agarose pad is used in combination with a cell-imaging dish, in which the predatory cells can move freely beneath the agarose pad while the immobilized prey cells are able to form bdelloplasts (Figure 2). This set-up is prepared based on specific strains of both E. coli and B. bacteriovorus, but the protocol may be easily altered to fit a user’s individual strains (e.g., carrying different selection markers, proteins fused with different fluorophores, etc.).

In this case, to visualize B. bacteriovorus during the attack phase, a specific strain (HD100 DnaN-mNeonGreen/PilZ-mCherry) was constructed that expresses a fluorescently tagged version of the cytoplasmic protein, PilZ (available in our laboratory upon request)⁷. This strain additionally produces DnaN (the β-sliding clamp), a subunit of DNA polymerase III holoenzyme, fused with a fluorescent protein. This enables ongoing DNA replication to be monitored inside the predatory cells as they grow within bdelloplasts.

Although the described protocol and software used for image acquisition refer to an inverted microscope provided by a specific manufacturer (see Table of Materials), this technique may be adjusted for any inverted microscope equipped with an environmental chamber or other external heating holder and capable of time-lapse imaging. For data analysis, users may choose any available software compatible with the individual output formats.

Figure 1: B. bacteriovorus life cycle in E. coli as a host cell. During the attack phase, a free-swimming B. bacteriovorus cell searches for and attaches to a host E. coli cell. After the invasion, the predatory cell becomes localized in the prey’s periplasm, changing the host cell’s shape and forming a bdelloplast. The reproductive phase starts with bdelloplast formation. The predatory cell digests the prey cell and reuses simple compounds to build its own structures. B. bacteriovorus grows as a long single filament inside the host’s periplasm. When the prey cell’s resources are exhausted, the B. bacteriovorus filament synchronously septates and forms progeny cells. After the progeny cells develop their flagella, they lyse the bdelloplast. Please click here to view a larger version of this figure.

Protocol

1. Preparation of B. bacteriovorus lysate for microscopic analysis

1. In a 250 mL flask, set up the coculture by combining 1 mL of fresh 24 h B. bacteriovorus culture (e.g., HD100 DnaN-mNeonGreen/PilZ-mCherry) and 3 mL of overnight prey culture (e.g., E. coli S17-1 pZMR100) with 50 mL of Ca-HEPES buffer (25 mM HEPES, 2 mM CaCl₂ [pH = 7.6]) supplemented with antibiotics when needed (here, 50 µg/mL kanamycin).
2. Incubate the coculture for 24 h at 30 °C with shaking at 200 rpm.
3. Check that the prey cells are fully lysed by liquid mounted phase-contrast microscopy. At this point, numerous motile attack phase B. bacteriovorus cells should be present, and no E. coli cell or bdelloplast should be visible.
4. Filter the B. bacteriovorus culture through a 0.45 µm pore size filter to remove any remaining host cells. Predatory cells (0.3–0.5 µm x 0.5–1.4 µm) freely penetrates 0.45 µm pores, whereas host cells are retained on the filter surface.
5. Spin down the filtrate for 20 min at 2469 x g and 30°C in a 50 mL conical tube to collect predatory cells. Resuspend the B. bacteriovorus pellet in 3 mL of Ca-HEPES buffer (to a final OD₆₀₀ of approximately 0.2) and incubate at 30 °C and 200 rpm for 30 min.

2. Preparation of host cells for B. bacteriovorus invasion

1. Use a single colony of the host strain (E. coli S17-1 pZMR100 is recommended due to various cell sizes resulting in the formation of unequally sized bdelloplasts) to inoculate 10 mL of YT medium (0.8% Bacto tryptone, 0.5% yeast extract, 0.5% NaCl [pH = 7.5]) that has been supplemented with antibiotics, if needed (here, 50 µg/mL kanamycin). Culture cells overnight at 37 °C and 180 rpm.
2. Transfer 2 mL of overnight E. coli culture (OD₆₀₀ of ~3.0) to a 2 mL test tube and centrifuge at room temperature (RT) and 2469 x g for 5 min. Resuspend the pellet in 200 µL of Ca-HEPES buffer.

3. Set-up of the microscope (see Table of Materials)

1. Pre-warm a microscopic chamber with a 100x oil immersion objective to 30 °C for at least 1 h before use. This step is required to prevent errors in maintaining focus during the experiment.
2. Turn on both the microscope and microscopy automation. Initialize the control software and select the appropriate filters to acquire brightfield/DIC/phase-contrast and fluorescence images, as needed (here: BF images, GFP/mCherry polychromic filter, and emission/excitation for mCherry and GFP).

4. Assembly of layouts for time-lapse fluorescence microscopy

1. Mix 200 mg of low fluorescent molecular grade agarose with 20 mL of Ca-HEPES buffer (1% final agarose concentration) and dissolve the agarose in a microwave. The batch can be reused several times after it solidifies, so simply repeat the heating step.
2. Pour 3 mL of melted agarose into a 35 mm glass-bottom dish (Table of Materials). Allow the agarose to solidify.
3. Using a laboratory micro-spatula, carefully remove the agarose pad from the 35 mm dish without disturbing the center pole of the agarose pad. Flip the pad bottom side facing up and place it in a Petri dish cover (Figure 2).
4. Put 5 µL of concentrated E. coli suspension on top of the flipped pad and spread cells in the center pole using an inoculation loop. Add 5 µL of concentrated B. bacteriovorus suspension (OD₆₀₀ of ~0.2) onto the E. coli-coated surface. Do not spread the cells.
5. Quickly return the agarose gel to the 35 mm glass-bottom dish (top side facing down), then cover with a lid. If necessary, remove any air bubbles by gently pressing down the agar against the plate.

Figure 2: Schematic depiction of the experimental workflow. The agarose pad is removed from the 35 mm dish and flipped so that the bottom side faces upwards. Fresh suspensions of predatory cells and overnight culture of prey cells are placed on the flipped pad to coat the “bottom” side. The agarose pad is then flipped to its original orientation and placed back in the 35 mm dish, which is mounted onto the inverted microscope stage in the microscope chamber. Please click here to view a larger version of this figure.

5. Conduction of time-lapse fluorescence microscopy

1. Place the 35 mm dish in the Petri dish holder such that it will not move during the course of the experiment. Place one drop of immersion oil (1.518 refractive index) onto the objective as well as the bottom of the 35 mm dish.
2. Mount the holder onto the stage of the inverted microscope in the microscope chamber.
3. Set up the options in the microscope control software to collect multiple images at multiple stage positions over time.
   1. Set the magnification (100x) and polychromic lens (GFP/mCherry). Using an eye piece and brightfield (BF), find the focal plane and open the point list manager.
   2. Select at least 10 positions of interest by moving the stage and storing the coordinates for each position in the microscope software by clicking the Mark point button. Avoid positions located close to each other to prevent photobleaching and phototoxicity. Turn the camera valve from the eyepiece to the monitor and calibrate each point by setting the focal plane and clicking the Replace point button in the point list manager.
   3. Open the experiment designer and set all experimental parameters in each tab as follows:
      1. Unmark the Z-stacking box.
      2. Choose fluorescent channels and set the optimal illumination settings. Here, the following settings were used: for the mCherry channel, mCherry filter sets (EX575/25; EM625/45), 50% intensity, and 200 ms exposure time; for the GFP channel, GFP filter set (EX475/28; EM525/48), 50% intensity, and 80 ms; and for the BF images, POL channel, 5% intensity, and 50 ms.
      3. Select intervals between acquiring images and the total time of the experiment. Here, 5 min intervals were performed over the 10 h period of the experiment.
      4. Enable focus maintenance for the chosen positions (for the system used here, by marking the Maintain focus with UltimateFocus check box).
      5. Select the data folder in which the image files should be automatically saved.
      6. Recheck all settings in the microscope software control, then start the time-lapse experiment.
4. After the first hour of the experiment, check that all stage positions are still in focus. Adjust the focus during time intervals between image acquisition, if needed.
5. When the time-lapse experiment is finished, remove the Petri dish and utilize the 35 mm dish with an agarose pad according to the biosafety protocol.
   NOTE: All substeps after step 5.2 are specific for DeltaVision Elite users. Researchers using other systems need to adjust the settings according to individual systems.

6. Processing of time-lapse images and generation of movies using Fiji software

1. Transfer experimental data to a computer loaded with the Fiji software. Launch the Fiji program and open an image using File | Open or by simply dragging and dropping the image file into Fiji.
2. In Bio-Formats Import Options, choose View stack with Hyperstack in the stack viewing options, Click on Composite color mode in the color options, and mark Autoscale.
3. By scrolling through the image stacks, assess whether the cells and bdelloplasts remained in focus over the course of the experiment. Check whether green fluorescent spots from DnaN-mNeonGreen are present within B. bacteriovorus cells inside bdelloplasts throughout the growth phase, and ensure that all stages of the predatory life cycle can be visualized.
4. To analyze a particular B. bacteriovorus cell/bdelloplast, mark it using the selection tools in the Fiji menu (Rectangle, Oval, Polygon, or Freehand). Duplicate the chosen region using Image | Duplicate or by using Ctrl + Shift + D.
5. Adjust the brightness and contrast for each fluorescence channel as follows:
   1. To choose a single channel, open Channel tools by clicking Image | Color | Channels tools or Ctrl + Shift + Z, and select the channel of interest (Channel 1: mCherry, Channel 2: mNeonGreen, Channel 3: brightfield).
   2. Adjust the brightness and contrast of images in the fluorescence channel by opening the B&C menu in Image | Adjust | Brightness/Contrast or by clicking Ctrl + Shift + C.
6. Add scale bar using Analyze | Tools | Scale bar. Add the time stamper to the images by clicking Images | Stacks | Time Stamper.
7. To save modified images, go to File | Save As and choose TIFF to save as an image sequence, AVI to produce a movie, or PNG to save a single image of the current frame.

Representative Results

The described TLFM-based system allows individual cells of B. bacteriovorus to be tracked in time (Figure 3, Movie 1) and provides valuable information about each stage of the complex predatory life cycle. The PilZ-mCherry fusion enables the entire predatory cell to be labeled in the attack phase as well as early stage of the growth phase (Figure 3). The transition from the attack to replicative phase was visualized not only by the host bdelloplast formation but also by the appearance of the replisome (replication machinery), which was marked by DnaN-mNeonGreen fluorescent foci (Figure 3).

As previously demonstrated, the replisome was assembled at the invading cell pole (pili-pole)⁷, and more than one replisome was observed within a growing B. bacteriovorus filament as the reproduction phase progressed (Figure 3). After several copies of the chromosome were synthesized, the replication was terminated (visualized as the disappearance of DnaN-mNeonGreen foci). Finally, the multinucleoid filament underwent septation, and the progeny cells were released from the bdelloplast (Figure 3).

The presented protocol describing the image processing using the Fiji software provides a step-by-step explanation of how to produce a movie for publication from the acquired time-lapse series (Movie 1).

Figure 3: B. bacteriovorus life cycle followed by time-lapse fluorescence microscopy. (A) Free-living predatory (B. bacteriovorus DnaN-mNeonGreen/PilZ-mCherry) and host (E. coli) cells. (B) Attachment of B. bacteriovorus için E. coli. (C) Bdelloplast formation. (D,E) Filamentous growth and chromosome replication. (F) Termination of chromosome replication. (G) Start of synchronous filament septation. (H) Bdelloplast lysis and release of progeny cells. Upper panels show brightfield images, lower panels represent merged brightfield and fluorescence channels. Scale bar = 1 µm, time = hh:min. Please click here to view a larger version of this figure.

Movie 1: Time-lapse imaging of B. bacteriovorus life cycle in E. coli as a host cell. Subcellular localization of DnaN-mNeonGreen (green) in strain HD100 DnaN-mNeonGreen/PilZ-mCherry. Red channel indicates labeling of the predator cells with cytoplasmic PilZ-mCherry. Grey indicates brightfield. Images were collected every 5 min. Please click here to download this video.

Discussion

Due to the increased interest in using B. bacteriovorus as a living antibiotic, new tools for observing the predatory life cycle, particularly predator-pathogen interactions, are needed. The presented protocol is used to track the entire B. bacteriovorus life cycle, especially during its growth inside the host, in real-time. Moreover, the application of a strain producing fluorescently tagged beta clamp of DNA polymerase III holoenzyme enabled monitoring of chromosome replication progression throughout the reproduction phase of B. bacteriovorus.

A critical step in this method is the proper preparation of layouts in the 35 mm dish. A challenge in observing the life cycle of B. bacteriovorus under the microscope is to establish conditions that support predatory growth (involving other gram-negative bacterial cells as host cells) and provide for predatory cell motility. One of the crucial aspects of effective microscopic observations is the need for a uniform distribution of prey on the agarose pad, which is achieved by spreading the cells with an inoculating loop. The host cell density cannot be too high, and the chosen observation points should contain a sufficient number of separate host cells. Meanwhile, the Bdellovibrio cells should be motile enough to actively search for their prey. Thus, they should not be spread out or air-dried before the pad is flipped back over and placed into the dish. Sometimes greater volumes of predator suspension are needed to provide sufficient motility in a thin layer of fluid between the agarose and glass surface.

A µ-Dish is used here; however, this is not essential for the protocol. Any glass-bottom dish could be used. An advantage of the µ-Dish is the optimal height and volume of the agarose path, which enables the coculture of predatory and host to be placed on the bottom side of the agarose pad. It is also important to use fresh B. bacteriovorus cells in the experiments, as the cells lose their motility during prolonged storage. A limitation of this protocol is that, at the single field of view, users can count approximately 100 predator cells and 100 host cells, but the MOI can be estimated at 50%–60%.

Current knowledge about the Bdellovibrio cell cycle is largely based on studies that have employed E. coli or P. aeruginosa. This system allows monitoring of B. bacteriovorus preying on a variety of bacterial pathogens, including Salmonella spp., S. flexneri, Proteus mirabilis, or K. pneumoniae. Moreover, this platform may be useful in experiments involving multidrug-resistant pathogens. Thus, it may help to facilitate improvements in the genetic engineering of B. bacteriovorus as a live antibiotic in human and veterinary medicine, particularly to combat multidrug-resistant pathogens.

Materials

Centrifuge	MPW MED. INSTRUMENTS	MPW-260R	Rotor ref. 12183
CertifiedMolecular Biology Agarose	BIO-RAD	161-3100	low fluorescence agarose for agarose pad
Fiji	ImageJ	https://imagej.net/Fiji	Open source image processing package
Glass Bottom Dish 35 mm	ibidi	81218-200	uncoated glass
Microscope	GE	DeltaVision Elite	Microtiter Stage, ultimate focus laser module, DV Elite CoolSnap HQ2 Camera, SSI assembly FP DV, kit obj. Oly 100x oil 1.4 NA, prism Nomarski 100x LWD DIC, ENV ctrl IX71 uTiter opaQ 240 V
Minisart Filter 0.45 µm	Sartorius	16555———-K	Cellulose Acetate, Sterile, Luer Lock Outlet
Start SoftWoRx	GE		Manufacturer-supplied imaging software