Name: live-cell imaging of the life cycle of bacterial predator bdellovibrio bacteriovorus using time-lapse fluorescence microscopy
Uploaded: 2020-05-08T14:00:00
Description: Watch this Scientific Journal Video about Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy at JoVE.com

This study was supported by the National Science Centre grant Opus 2018/29/B/NZ6/00539 to J.Z.C.

1. Preparation of B. bacteriovorus lysate for microscopic analysis
<ol>
	<li>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 &#181;g/mL kanamycin).</li>
	<li>Incubate the coculture for 24 h at 30 &#176;C with s...

<ol>
	<li>Shatzkes, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predatory+Bacteria+Attenuate+Klebsiella+pneumoniae+Burden+in+Rat+Lungs.">Predatory Bacteria Attenuate Klebsiella pneumoniae Burden in Rat Lungs.</a> mBio. 7 (6), (2016).</li><li>Iebba, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bdellovibrio+bacteriovorus+directly+attacks+Pseudomonas+aeruginosa+and+Staphylococcus+aureus+Cystic+fibrosis+isolates.">Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates.</a> Frontiers in Microbiology. 5, (2014).</li><li>Willis, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injections+of+Predatory+Bacteria+Work+Alongside+Host+Immune+Cells+to+Treat+Shigella+Infection+in+Zebrafish+Larvae.">Injections of Predatory Bacteria Work Alongside Host Immune Cells to Treat Shigella Infection in Zebrafish Larvae.</a> Current biology: CB. 26 (24), 3343-3351 (2016).</li><li>Lambert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+flagellar+filament+and+the+role+of+motility+in+bacterial+prey-penetration+by+Bdellovibrio+bacteriovorus.">Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus.</a> Molecular Microbiology. 60 (2), 274-286 (2006).</li><li>Lambert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Predatory+Patchwork:+Membrane+and+Surface+Structures+of+Bdellovibrio+bacteriovorus.">A Predatory Patchwork: Membrane and Surface Structures of Bdellovibrio bacteriovorus.</a> Advances in Microbial Physiology. 54, 313-361 (2008).</li><li>Makowski, &. #. 3. 2. 1. ;., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+Chromosomal+Replication+in+Predatory+Bacterium+Bdellovibrio+bacteriovorus.">Initiation of Chromosomal Replication in Predatory Bacterium Bdellovibrio bacteriovorus.</a> Frontiers in Microbiology. 7, 1898 (2016).</li><li>Makowski, &. #. 3. 2. 1. ;., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+Chromosome+Replication+and+Its+Relationship+to+Predatory+Attack+Lifestyles+in+Bdellovibrio+bacteriovorus.">Dynamics of Chromosome Replication and Its Relationship to Predatory Attack Lifestyles in Bdellovibrio bacteriovorus.</a> Applied and Environmental Microbiology. 85 (14), (2019).</li><li>Fenton, A. K., Kanna, M., Woods, R. D., Aizawa, S. I., Sockett, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shadowing+the+actions+of+a+predator:+backlit+fluorescent+microscopy+reveals+synchronous+nonbinary+septation+of+predatory+Bdellovibrio+inside+prey+and+exit+through+discrete+bdelloplast+pores.">Shadowing the actions of a predator: backlit fluorescent microscopy reveals synchronous nonbinary septation of predatory Bdellovibrio inside prey and exit through discrete bdelloplast pores.</a> Journal of Bacteriology. 192 (24), 6329-6335 (2010).</li><li>Kuru, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+D-amino-acids+reveal+bi-cellular+cell+wall+modifications+important+for+Bdellovibrio+bacteriovorus+predation.">Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation.</a> Nature Microbiology. 2 (12), 1648-1657 (2017).</li><li>Dashiff, A., Junka, R. A., Libera, M., Kadouri, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predation+of+human+pathogens+by+the+predatory+bacteria+Micavibrio+aeruginosavorus+and+Bdellovibrio+bacteriovorus.">Predation of human pathogens by the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus.</a> Journal of Applied Microbiology. 110 (2), 431-444 (2011).</li></ol>

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 ...

Bdellovibrio bacteriovorus is a small (0.3&#8211;0.5 &#181;m by 0.5&#8211;1.4 &#181;m) gram-negative bacterium that preys on other gram-negative bacteria, including harmful pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and Shigella flexneri1,2,3. 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 &#181;m/s, searches for its prey. After attaching to the prey&#8217;s outer membrane, it enters the periplasm4,5. During the interperiplasmic reproductive phase, B. bacteriovorus uses a plethora of hydrolytic enzymes to degrade the host&#8217;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 analysis2,9,10, 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&#8217;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 &#946;-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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61105/61105fig01.jpg" /> 
Figure 1: B. bacteriovorus life cycle in E. coli as a host cell.&#160;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&#8217;s periplasm, changing the host cell&#8217;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&#8217;s periplasm. When the prey cell&#8217;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. <a href="https://www.jove.com/files/ftp_upload/61105/61105fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>Centrifuge</td>
			<td>MPW MED. INSTRUMENTS</td>
			<td>MPW-260R</td>
			<td>Rotor ref. 12183</td>
		</tr>
		<tr>
			<td>CertifiedMolecular Biology Agarose</td>
			<td>BIO-RAD</td>
			<td>161-3100</td>
			<td>low fluorescence agarose for agarose pad</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>ImageJ</td>
			<td>https://imagej.net/Fiji</td>
			<td>Open source image processing package</td>
		</tr>
		<tr>
			<td>Glass Bottom Dish 35 mm</td>
			<td>ibidi</td>
			<td>81218-200</td>
			<td>uncoated glass</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>GE</td>
			<td>DeltaVision Elite</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Minisart Filter 0.45 &#181;m</td>
			<td>Sartorius</td>
			<td>16555----------K</td>
			<td>Cellulose Acetate, Sterile, Luer Lock Outlet</td>
		</tr>
		<tr>
			<td>Start SoftWoRx</td>
			<td>GE</td>
			<td></td>
			<td>Manufacturer-supplied imaging software</td>
		</tr>
	</tbody>
</table>

live-cell imaging of the life cycle of bacterial predator bdellovibrio bacteriovorus using time-lapse fluorescence microscopy

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 &#946;-subunit of DNA polymerase III further allows chromosome replication to be monitored during the reproduction phase of the B. bacteriovorus life cycle.

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...

Watch this Scientific Journal Video about Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy at JoVE.com

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

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.

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

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...

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