Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Summary

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 cycl....

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 att....

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.......

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 .......

Materials

Name	Company	Catalog Number	Comments
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