Name: induction of cellular differentiation and single cell imaging of vibrio parahaemolyticus swimmer and swarmer cells
Uploaded: 2017-05-15T15:00:00
Description: Watch this Scientific Journal Video about Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells at JoVE.com

This work was supported by the Max Planck Society.

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.
<ol>
	<li>Streak out V. parahaemolyticus cells from a -80 &#176;C glycerol stock onto a fresh LB ag...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+ParA-like+ATPases+promotes+cell+pole+maturation+by+facilitating+polar+localization+of+chemotaxis+proteins.">A family of ParA-like ATPases promotes cell pole maturation by facilitating polar localization of chemotaxis proteins.</a> Genes Dev. 25, 1544-1555 (2011).</li><li>Vischer, N. O. 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=Cell+age+dependent+concentration+of+Escherichia+coli+divisome+proteins+analyzed+with+ImageJ+and+ObjectJ.">Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.</a> Front Microbiol. 6 (JUN), (2015).</li><li>Schneider, C. a., Rasband, W. S., Eliceiri, K. 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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 analysis14,15,16,17,18, the analysis of...

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 components3,4,5. 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&#180;-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 &#8211; 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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB-medium</td>
			<td>Roth</td>
			<td>X968.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Agar, granulated</td>
			<td>BD</td>
			<td>214510</td>
			<td>For preparation of LB agar with LB-medium</td>
		</tr>
		<tr>
			<td>Difco Heart Infusion Agar</td>
			<td>BD</td>
			<td>244400</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Agarose NEEO, ultra quality</td>
			<td>Roth</td>
			<td>2267.3</td>
			<td>For preparation of microscopy agarose pads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Additives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-arabinose</td>
			<td>Roth</td>
			<td>5118.3</td>
			<td>Used to induce pBAD derivatives</td>
		</tr>
		<tr>
			<td>2,2`-Bipyridyl</td>
			<td>Sigma Aldrich</td>
			<td>D216305-25G</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Roth</td>
			<td>5239.1</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Roth</td>
			<td>K029.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Roth</td>
			<td>3886.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>-</td>
			<td>-</td>
			<td>Standard recipe for Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>D(+) Sucrose</td>
			<td>Roth</td>
			<td>4621.1</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Roth</td>
			<td>3783.5</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Hardware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 150x20mm with cams</td>
			<td>Sarstedt</td>
			<td>82.1184.500</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>kelvitron t (B 6420)</td>
			<td>Heraeus</td>
			<td>-</td>
			<td>Used for drying HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Microbial System</td>
			<td>BioRad</td>
			<td>1652662</td>
			<td>Used for elctroporation of plasmid DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph Offline (version 7.7.5.0)</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>Used for microscopy image analysis</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>Used for microscopy data analsis</td>
		</tr>
	</tbody>
</table>

induction of cellular differentiation and single cell imaging of vibrio parahaemolyticus swimmer and swarmer cells

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.

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...

Watch this Scientific Journal Video about Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells at JoVE.com

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

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.

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

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

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